title: fyn_permissivity_output3

city: Barcelona

first: Albert

last: Escobedo

state: Catalonia

address: Carrer Dr Aiguader 88

department: Systems and synthetic biology

country: Spain

institute: CRG

data_processing:
DiMSum (Demultiplex with Cutadapt)
DiMSum (QC raw reads with FastQC)
DiMSum (Trim reads with Cutadapt)
DiMSum (Process and analyse variants with DiMSum)
Assembly: NA
Supplementary files format and content: All in tabular format. sparse_DTS_core_mutagenesis_scores.csv contains a list of variants, each linked to the protein they are related to (Protein), its Uniprot identifier (UniprotCode), their complete amino acid sequence (aa_seq), the sequence in core positions (core), the hamming distance to the WT protein they are related to (Nham_aa), whether they were used as the reference in DiMSUM processing (WT), and their fitness and error values as obtained directly from DiMSum (fitness_raw and sigma_raw) and after scaling in the 0-1 range (fitness_scaled and sigma_scaled). The FYN-SH3_core_DTS_binding_scores_PDR1super.csv file contains equivalent aa_seq, core, Nham_aa, WT, fitness_scaled, sigma_scaled, fitness_raw and sigma_raw fields for variants all related to FYN SH3 binding to PDR1super. The FYN-SH3_core_permissivity_scores contains equivalent aa_seq, WT, fitness_scaled, sigma_scaled, fitness_raw and sigma_raw fields for variants in the permissivity library.
Library strategy: PCR amplicon

source_name: yeast cells

growth_protocol: The bottlenecked library pool containing randomly subsampled fractions of the FYN SH3, CI-2A and CspA DTS core libraries in pGJJ162, the full complexity FYN SH3 DTS core library in pGJJ159-PRD1super and the FYN SH3 permissivity library in pGJJ162 were transformed in BY4741 yeast cells by heat shock. The number of transformation reactions was scaled depending on the final selection volume, which was in turn scaled to achieve a 100X coverage at the most stringent selection step in terms of cell count bottlenecking (inoculation in selection medium, see below). An individual transformation was performed per 200 mL of selection culture. For each individual transformation, 25 mL YPDA were inoculated with BY4741 colonies from a freshly streaked YPDA plate and grown overnight at 30ºC. The process was performed in parallel for each independent biological replicate, starting from a different BY4741 colony (2 replicates for the subsample DTS core library pool, 3 replicates for the other experiments). The precultures were used to inoculate 180 mL of YPDA to an OD600 of 0.3, which was grown for 4 h at 30ºC. Cells were harvested by 5 min centrifugation at 3,000 g, washed in water and treated with 8.6 mL SORB for 30 min. 3.5 μg of plasmid DNA were then added along with 175 μL of 10 mg/mL sonicated salmon sperm DNA (Santa Clara, CA, US), and the mixture was agitated at room temperature for 10 minutes followed by addition of 35 mL of plate mixture plus another 30 min incubation. 3.5 mL of DMSO were then added and the mixture was heat-shocked at 42ºC for 20 min. After the heat-shock, cells were harvested by 5 min centrifugation at 3,000 g, resuspended in recovery media and incubated at 30ºC for 1 h. Cells were then harvested again and resuspended in 200 mL of SC -URA selective medium. A 10 μL aliquot was serially diluted and plated in SC -URA to assess transformation efficiency.  All biological replicate transformation cultures were grown in parallel at 30ºC for ~24 h. Then, the selection input cultures were inoculated by resuspending water washed harvested cell pellets in SC -URA -ADE to an OD600 of 0.1 (800 mL in 5 L flasks in the cases of the subsampled DTS core library pool and FYN SH3 binding library, 400 mL in 2L flasks in the case of the FYN SH3 permissivity library). The input cultures were grown at 30ºC and harvested upon reaching an OD600 of 1.6 (5 generations), which typically occurred from 12 to 15 h after inoculation. Cell pellets were stored at -20ºC. Aliquots of the input culture were used to inoculate the selection (output) cultures (SC -URA -ADE, 200 μg/mL methotrexate) at an OD600 of 0.05. The output cultures were grown at 30ºC for ~30 h (subsampled DTS core library pool and FYN SH3 binding library) or until they reached an OD600 of 1.6. At that time, cells were harvested by 5 min centrifugation at 3,000 g, thoroughly washed in water and stored at -20ºC.

extract_protocol:
Input and output culture cell pellets were resuspended in DNA extraction buffer (10 mM Tris-HCl, 100 mM NaCl, 2% Triton-X, 1% SDS, pH 8). Volumes were scaled depending on the volume of cell pellets to process. For pellets from 100 mL cultures harvested at OD600 1.6, 1 mL of DNA extraction buffer was used. Cells were lysed by cyclically freezing in liquid nitrogen followed by incubation in a 62ºC water bath twice. Total DNA was extracted by adding 1 mL of a 25:24:1 phenol:chloroform:isoamyl alcohol mixture (Merck, Darmstadt, Germany) along with 1 g of acid washed glass beads (Sigma-Aldrich, Saint Louis, MO, US) and vortexing for 10 min. The mixture was centrifuged at room temperature for 30 min at 3,300 g and the aqueous phase (upper layer) was recovered in a separate tube. 1 mL of the phenol:chloroform:isoamyl alcohol was added to it and the mixture was vortexed for 2 min followed by 45 min centrifugation at 3,300 g. The aqueous phase was again transferred to a new tube and mixed with 0.1 volumes of 3 M sodium acetate and 2.2 volumes of pure ethanol previously cooled down at -80ºC. The mixture was kept at -20 ºC for 30 min and spun down at 3,300 g for 30 min at 4ºC. The DNA pellets were dried overnight in a fume extraction hood.  Plasmid DNA from total DNA extracts was quantified by qPCR using the oGJJ152-oGJJ153 oligo pair that anneals at the origin of replication of all pGJJ vector series. The qPCR reaction was performed in a LightCycler 400 instrument (Roche, Basel, Switzerland) using the SYBR green qPCR 2X master mix from Thermo Fisher (Waltham, MA, US). Quantification was achieved by measuring a standard curve on a serial dilution of a control sample of known concentration in the same qPCR run as the query samples, and subtracting blank measurements.  Library preparation for sequencing in Illumina instruments followed a two-step PCR protocol. Frameshifts and a segment of the Illumina adapter overhangs were introduced in PCR1. For each sample, 8x50 μL PCR1 reactions were performed using 31.25 million plasmid molecules as a template as quantified by qPCR. PCR1 used the oGJJ595 and the oGJJ748 frameshifting oligo pools and consisted of 11 cycles. Next, all reactions corresponding to the same sample were pooled and treated with 16 μL ExoSAP for 30 min at 37ºC, then for 20 min at 80ºC. Samples were cleaned-up using MinElute columns, eluted in 30 μL of pre-warmed water (55ºC) and 1.5 μL were used as template for each PCR2 reaction.16x50 μL PCR2 reactions were performed per sample to introduce the rest of the Illumina adapters and the dual index barcodes. PCR2 reactions corresponding to the same sample were pooled, concentrated using a MinElute column, and run in a 2% agarose gel. Bands of the correct sizes were excised from the gel and sequencing-ready DNA was recovered by using a centrifugal filter unit followed by MinElute clean-up. Samples were quality controlled by the CRG Genomics core facility using a TapeStation instrument (Agilent, Santa Clara, CA, US), quantified by qPCR and sequenced in either a NextSeq500 (FYN SH3 permissivity library) or NextSeq2000 (subsampled core DTS library pool and FYN SH3 binding library) instrument (Illumina, San Diego, CA, US).
Combinatorial core libraries using a reduced alphabet of hydrophobic amino acids (F, L, I, M, V) were built by multiple segment Gibson assembly of oligonucleotides bearing the DTS degenerate codon at core residue-encoding positions (oligos used in this study were all obtained from IDT, Coralville, IA, US). Each of the genes encoding the FYN SH3 domain, CI-2A and CspA were codon optimized for expression in Saccharomyces cerevisiae and divided in two segments smaller than 200 bp each, which was the size limit for commercial synthetic degenerate oligo production at the time of performing this study. Both the 5’ and the 3’ gene segments contained a homology region of at least 25 bp to the linearized pGJJ191 mutagenesis vector, plus an internal homology region of at least 25 bp. Double stranded Gibson assembly inserts were produced from single stranded commercial oligonucleotides via a single cycle PCR reaction using a 3’ annealing reverse primer specific to each segment (see Table ). The pGJJ191 vector was linearized via a 25-cycle PCR reaction using primers oGJJ311 and oGJJ406, followed by 1h DpnI (NEB) treatment at 37ºC plus inactivation at 80ºC for 20 minutes (1 μL enzyme per 50 μL PCR reaction). Both inserts and vector PCR reactions were cleaned-up in MinElute columns (Qiagen, Hilden, Germany). Gibson assembly mixtures contained 300 ng of linearized pGJJ191 plus the two insert segments at a 5:1 molar ratio each, along with a Gibson enzyme mixture produced in house by the Protein Technologies core facility at the CRG.  Conversely, the FYN SH3 permissivity library was purchased as an oligo pool from Twist Bioscience (San Francisco, CA, US). The oligo pool was amplified in a 13 cycle PCR reaction with primers oAE018 and oAE227, which was cleaned up using a MinElute column. The insert amplified oligo pool contained 25 bp homology regions at both ends and was mixed at a 5 to 1 molar ratio with 300 ng of linearized pGJJ191 and subsequently with the Gibson enzyme mixture.  All Gibson assembly reactions were incubated at 50ºC for 8h, dialyzed against water using 0.025 μm filters (Millipore, Burlington, MA, US) for 1.5h, concentrated using a SpeedVac and transformed into E. coli C3020 cells (NEB, Ipswich, MA, US). The number of transformants was assessed by colony count in serial dilutions of transformation outgrowth aliquots in LB plates supplemented with 50 μg/mL spectinomycin, and 1 to 5 equivalent transformation reactions were pooled to reach at least 20X variant coverage per library as required. Plasmid DNA was extracted using the Qiagen Plasmid Plus Midi kit and the library inserts were obtained through double endonuclease digestion (HindIII-HF and NheI-HF, NEB) followed by agarose gel purification achieved by a combined use of centrifugal filter units (Millipore) and MinElute clean-up. Inserts were introduced in double-restricted, Quick CIP-trated (NEB) pGJJ162 (abundancePCA) or pGJJ159-PRD1super (bindingPCA) vectors by overnight thermal-cyclic ligation using T4 DNA ligase. The pGJJ159-PRD1super vector was previously obtained by introducing the S. cerevisiae codon-optimized PRD1super encoding gene from commercial oligo synthesis (IDT) into BamHI-SpeI double restriction-linearized pGJJ159 via Gibson assembly. Ligation mixtures were dialyzed against water, SpeedVac-concentrated and transformed into E. coli C3020 electrocompetent cells. Equivalent transformations were combined to reach at least 20X variant coverage, and selection-ready libraries in PCA vectors were Midi-prep extracted.  For sparse sampling of combinatorial core libraries (FYN SH3, CI-2A and CspA in pGJJ162), the full-complexity libraries were transformed into E. coli C3020 electrocompetent cells and bottlenecked by outgrowth medium serial dilution into overnight selective medium (LB supplemented with 50 μg/mL ampicillin) assessed in parallel through colony count of plated aliquots, aiming at ~10,000 transformants per library. Bottlenecked libraries were extracted by mini-prep, qPCR quantified and mixed to equivalent molar ratios.

treatment_protocol: abundancePCA and bindingPCA (High-resolution mapping of protein concentration reveals principles of proteome architecture and adaptation, Levy and Michnick, 2014; Mapping the energetic and allosteric landscapes of protein binding domains, Faure et. Al, Nature 2022)

tag: lab strain

value: yeast cells


title: S7: Meningoma

status: Public on May 13 2024

type: SRA

city: Guangzhou

first: by

last: h

address: Guangzhou, Guangdong Province

country: China

institute: Jinan University

library_source: transcriptomic single cell

data_processing:
The Cell Ranger takes FASTQ files and performs alignment, filtering, barcode counting, and UMI counting
The Seurat analyses data
Assembly: GRCh38
Supplementary files format and content: Matrix table with raw gene counts for every gene and every cell

molecule: polyA RNA

organism: Homo sapiens

source_name: Meningoma

extract_protocol:
The isolation of single cells was conducted in a sterile environment with the instruments pre-autoclaved and the solutions filtered through a 0.22μm filter. The fresh human meningioma tissues were put in cold L-isolation buffer (DPBS, 0.9mm CaCl2, 0.49mm MgCl2, 25mm D-glucose, then 1mm Sodium pyruvate), cut into small blocks of 3–5mm3 with a surgical blade, and placed after upside-down using pre-cooled PBS in a 50ml centrifuge tube. After most of the lumps sank, the supernatant containing a large number of cells related to the hematological system was discarded and repeated three to five times to obtain a relatively pure meningioma tissue mass. The tissue masses were placed in Petri dishes, minced as much as possible with a surgical blade, and transferred to a pre-cooled L-isolation buffer, where Dounce Tissue Grinder (Wheaton) was used to further mill the tissue masses into tissue suspension. The suspension was centrifuged at 2000g for 10 minutes and resuspended in a WASH buffer (L-isolation buffer containing 10% BSA). A 100μm cell screen was used for filtration to remove the uncrushed lumpy tissue and the filtrate was further filtered through a 40μm cell screen (Falcon) to obtain a crude cell suspension. After the crude suspension was washed twice with pre-cooled HBSS containing calcium and magnesium, the precipitate was resuspended in Digesting Buffer I (HBSS, 4%Dispase, 1mg/mL Collagen ASE I, 1mg/mL Collagen ASE II, 1mg/mL Collagen ASE IV, 5μg/mL DNA ASE I) and digested slowly in a rotating oven at 37°C for 1h. After digestion, the samples were centrifuged at 500g for 5 minutes at low temperature, and the cell pellets were washed twice with HBSS and re-suspended in Digest Buffer II (HBSS, Neurological Isolation Enzyme, 5μg/mL DNA ASE I) for digestion by slow rotation in an incubator at 37°C for 1h. Once the digestion was complete, the filtrate was filtered through a 15μm cell screen (Pluriselect) and the filtrate became the single-cell suspension. After low-temperature centrifugation, the single-cell suspension was re-suspended in L-isolation buffer, and then every 700g/μl of suspension was slowly added to 1.6ml of L-isolation buffer containing 20%percoll, and the suspension was centrifuged at 0, 700g for 10 min to remove tissue debris and dead cells. The cell precipitate was directly resuspended in erythrocyte lysis buffer, treated at normal temperature for 10 minutes, washed with PBS twice, and then re-suspended in pre-cooled PBS. The obtained meningioma single cells were used for single-cell sequencing. For the primary culture of meningioma cells, the tissues were directly cut into pieces, added to a DMEM medium containing 10mg/ml Collagenase I and 5%FBS, and placed in an incubator at 37°C for digestion for 1h, during which the tissues were gently blown several times every 10 minutes using a Pap pipette. The digested cells were stripped of red blood cells with red blood cell lysates, washed twice with PBS, and resuspended in DMEM/F12 (3: 1) medium containing 10%FBS(Gbico), 10% HS (Horse serum) and 1% p/s. Cells were incubated at 37°C with 5% CO2 and the medium was changed every three days.
According to the manufacturer’s instructions, single-cell libraries were generated via the Chromium Controller and the Single Cell Reagent Kit 3’ v3 (10x Genomics, Pleasanton, CA),RNA libraries were prepared for sequencing using standard Illumina protocols

tag: tissue

value: Meningoma

tag: cell type

value: tumor

tag: grade

value: grade I


title: fyn_permissivity_output2

library_source: genomic

molecule: genomic DNA

organism: Saccharomyces cerevisiae


title: Asi_DMSO2_D2

city: salt lake city

first: Mahesh

middle: Babu

last: Chandrasekharan

state: Utah

address: 2000 circle of hope room 3715

department: Radiation Oncology

country: USA

institute: University of Utah SOM

description:
Asi_DMSO.bw
Asi_DMSO_N_peaks.narrowPeak

data_processing:
Adaptor and low-quality sequences were trimmed from paired-end sequencing reads using Trim Galore v 0.5.0
Bowtie2 (Langmead et al. 2012) was applied on trimmed read pairs to map reference genome (hg38) with the following setup: --dovetail –no-unal –no-mixed –no-discordant –very-sensitive -I 0 -X 1000.
After getting sam alignment files from Bowtie2, the files were converted to bam files using “samtools view -S -b” (Li et al. 2015). Bamfiles  were further purified by removing PCR duplicates using following settings: “picard MarkDuplicates REMOVE_DUPLICATES=true” and mitochondrial reads (http://broadinstitute.github.io/picard/).
Peak calling was used applying MACS2 (Zhang et al. 2008) using following setting: Macs2 call peak -f BAMPE -m 4 100 –bdg –SPMR (for narrow peak).
Bigwigs were generated using bins using deeptools bamCoverage with the following parameters: --normalizeUsing RPKM
Assembly: Hg38
Supplementary files format and content: Coverage in bigWig format
Supplementary files format and content: peak calls in narrowPeak
Library strategy: NicE-seq

source_name: Brain

growth_protocol: MTU87 and LTAU87 cells were cultured in DMEM supplemented with 10% Fetal bovine serum and 1% penicillin-streptomycin media.

extract_protocol:
Cells were fixed with 1% Formaldehyde
For library construction, MTU87 or LTAU87 cells with or without belinostat treatment were cross-linked using 1% formaldehyde for 10 min at room temperature and quenched by using 125 mM glycine. Nuclei were isolated by incubating the cross-linked cells in cytosolic buffer (15 mM Tris-HCl pH 7.5, 5 mM MgCl2, 60 mM KCl, 0.5 mM DTT, 15 mM NaCl, 300 mM sucrose, and 1% NP40) for 10 min on ice with occasional agitation. Nuclei were precipitated by spinning at 1000 × g, 4 °C for 5 min and the supernatant was discarded. Open chromatin DNA was labeled with biotin by incubating the nuclei in the presence of 2.5 U of Nt.CviPII (NEB R0626S), 10 U of DNA polymerase I (M0209S) and 30 μM of each dNTP including 6 μM of biotin-14-dATP (Invitrogen, 19524-016) and 6 μM of biotin-16-dCTP (ChemCyte, CC-6003-1) in 200 μL of 1× NEB buffer 2. The labeling reaction was carried out at 37 °C in a thermo-mixer at 800 RPM for 2 h. We added 20 μL of 0.5 M EDTA and 2 μg of RNase A to the labeling reaction and incubated it at 37 °C for 0.5 h to stop the reaction and digest RNA. Finally DNA–protein cross-linking was reversed by adding 20 μL of proteinase K (NEB, P8107S) and 20 μL of 20% SDS to the reaction and incubating at 65 °C for overnight. Biotin-labeled genomic DNA was extracted using phenol chloroform.                Biotin-labeled genomic DNA were sonicated into 150-bp fragments (Covaris) and 1 μg of DNA was end-repaired, dA-tailed, and ligated with NEB Illumina adaptor (E7370S). Without further purification, the ligation product was mixed with 50 μL of Streptavidin magnetic beads (Invitrogen 65001, blocked using 0.1% cold fish gelatin in 1× PBS overnight at 4 °C) in 1 mL of B&W buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 M NaCl). Biotin-labeled open chromatin DNA was captured by streptavidin magnetic beads at 4 °C for 2 h with end-over-end rotation. The beads were washed four times with B&W buffer plus 0.05% of Triton X-100 followed by one wash with TE plus Triton X-100. The beads were resuspended in 40 μL of nuclease-free water and 4 μL was used for library amplification using PCR (NEB, E7370S). Ten PCR cycles were usually sufficient to generate the amount of library DNA needed for sequencing.

treatment_protocol: MTU87 and LTAU87 cells were treated with 1uM belinostat for 24hr.

value: Brain

tag: cell line

value: Glioblastoma

tag: treatment


title: Asi_DMSO2_D1

description:
Asi_DMSO.bw
Asi_DMSO_N_peaks.narrowPeak


title: WSB_6_ORSAM11442_GT20-13807_heart

city: Bar Harbor

first: Gary

last: Churchill

state: Maine

address: 600 Main St

institute: The Jackson Laboratory

library_source: transcriptomic

data_processing:
Each tissue directory has a raw counts matrix, a vst matrix and mrna annotations table.  counts_matrix.csv: raw counts quantified by EMASE and processed by/RNAseq/Raw/GBRS/QTLViewer_setup/gathering_GBRS_total_counts.R script.  vst_matrix.csv: counts normalized by DESeq2 vst() function (quick version of variance stabilizing transformation). Matrix generated by /RNAseq/Raw/GBRS/QTLViewer_setup/QTLviewer_format.R script.  mrna_annots.csv: gene annotation file ENSEMBL version 75.
Assembly: GRCm38
Supplementary files format and content: counts_matrix.csv
Supplementary files format and content: mrna_annots.csv
Supplementary files format and content: vst_matrix.csv

molecule: total RNA

organism: Mus musculus

source_name: heart

extract_protocol:
RNA was isolated from tissue using the MagMAX mirVana Total RNA Isolation Kit (ThermoFisher) and the KingFisher Flex purification system (ThermoFisher). Frozen tissues were pulverized using a Bessman Tissue Pulverizer (Spectrum Chemical) and homogenized in TRIzol (ThermoFisher Scientific) using a gentleMACS dissociator (Miltenyi Biotec Inc). After the addition of chloroform to the TRIzol homogenate, the RNA-containing aqueous layer was removed for RNA isolation, according to the manufacturer’s protocol, beginning with the RNA bead binding step.
2 ul of diluted (1:1000) ERCC Spike-in Control Mix 1 (Ambion by Life Technologies) was added to 100ng of each RNA sample prior to library construction. Libraries were constructed using the KAPA mRNA HyperPrep Kit (Roche Sequencing and Life Science), according to the manufacturer’s protocol. Briefly, the protocol entails isolation of polyA containing mRNA using oligo-dT magnetic beads, RNA fragmentation, first and second strand cDNA synthesis, ligation of Illumina-specific adapters containing a unique barcode sequence for each library, and PCR amplification. The quality and concentration of the libraries were assessed using the D5000 ScreenTape (Agilent Technologies) and Qubit dsDNA HS Assay (ThermoFisher), respectively, according to the manufacturers’ instructions.

treatment_protocol: Mice were sensitized on a high-fat, high-sugar (HFHS) diet

value: heart

tag: diet

value: 45percent fat


title: S4: Meningoma


title: fyn_permissivity_output1


title: Asi_DMSO1_D2

description:
Asi_DMSO.bw
Asi_DMSO_N_peaks.narrowPeak


title: WSB_7_ORSAM11443_GT20-13781_heart


title: S3: Meningoma


title: fyn_permissivity_input3


title: Asi_DMSO1_D1

description:
Asi_DMSO.bw
Asi_DMSO_N_peaks.narrowPeak


title: WSB_5_ORSAM11441_GT20-13837_heart


title: WSB_4_ORSAM11440_GT20-13778_heart


title: S2: Meninges

source_name: Meninges

value: Meninges


title: S1: Meninges


title: fyn_permissivity_input2


title: fyn_permissivity_input1


title: Asi_Bel2_D2

description:
Asi_bel.bw
Asi_Bel_N_peaks.narrowPeak


title: Asi_Bel2_D1

description:
Asi_bel.bw
Asi_Bel_N_peaks.narrowPeak


title: S6: Meningoma


title: S5: Meningoma


title: WSB_1_ORSAM11438_GT20-13784_heart


title: PWK_9_ORSAM11437_GT20-13834_heart


title: Bari + IFNG, scRNAseq

city: Ann Arbor

first: Jennifer

last: Harder

state: MI

address: 1301 Catherine St.

department: Internal Medicine, Division of Nephrology

institute: University of Michigan Medical School

description:
10X Genomics
Sample12

data_processing:
Samples 1-8 (Bulk): Fastq files were mapped to ENSEMBL 38.104 using STAR 2.7.8a (PMID: 23104886)
Samples 1-8 (Bulk): Read counts were extracted from the fastq files using HTSeq (version0.11) (PMID: 25260700), voom-transformed (PMID: 24485249) and log-2 transformed.
Samples 9-12 (sc): The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v3.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: hg38/GRCh38
Supplementary files format and content: (Bulk)  bulkRNAseq_CountsGt0_voom_filtered_idmap.txt
Supplementary files format and content: (sc)  barcodes.tsv, features.tsv, matrix.mtx

source_name: Kidney organoid

extract_protocol:
Samples 1-8 (Bulk): Whole wells of D24 treated kidney organoids were lysed and RNA was extracted. Samples 9-12 (sc): Cells were dissociated from D24 treated kidney organoids using cold active protease into single cells.
Samples 1-8 (Bulk): Library prep and next-generation sequencing of total RNA on a NovaSeq (150 bp, paired end) were carried out in the Advanced Genomics Core at the University of Michigan. Samples 9-12 (sc): 10x Genomics sequencing was performed in the Advanced Genomics Core at the University of Michigan.

value: Kidney organoid

value: 1uM Bari + 25ng/ml IFNg


title: PWK_7_ORSAM11436_GT20-13811_heart


title: IFNG, scRNAseq

description:
10X Genomics
Sample11

value: 25ng/ml IFNg


title: PWK_6_ORSAM11435_GT20-13815_heart


title: D4H5 [20_S20]

city: Hannover

first: Stephan

last: Immenschuh

state: Deutschland

address: Carl-Neuberg-Str. 1

department: Institute for Transfusion Medicine and Transplant Engineering

country: Germany

institute: Hannover Medical School

data_processing:
Sequencing was performed at the Genomics service (ISCIII) on a NextSeq 500 sequencer using 75 base read lengths in paired-end mode.
First quality control analysis was based on fastQC v0.11.3 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and any adapter sequences and low quality 3′ ends were removed using Trimmomatic v0.36.
High-quality reads were mapped against Hg38 human genome using STAR v2.4.0.1 and mapping quality control was performed using RseQC v2.6.4.
Transcriptome prediction and gene/isoform quantification was calculated using HT-seq v0.11.1 based on Hg38 RefSeq reference genes
Normalization and differential expression analysis were performed on the raw counts using the R package DESeq2 v1.32.0.33
Assembly: Hg38
Supplementary files format and content: Raw counts table

source_name: human monocyte-derived macrophages (hMDMs)

extract_protocol:
RNA isolation was performed with an RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s recommendations
TruSeq Stranded mRNA Kit (Illumina) was used for library preparation based on the recommendations from the manufacturer.

value: Heme (lower concertration)


title: Bari, scRNAseq

description:
10X Genomics
Sample10

value: 1uM Bari for 1hr


title: D4H10 [18_S18]

value: human monocyte-derived macrophages (hMDMs)

value: Heme


title: PWK_5_ORSAM11434_GT20-13806_heart


title: Asi_Bel1_D1

description:
Asi_bel.bw
Asi_Bel_N_peaks.narrowPeak


title: THP1, UL138stopvirus, differentiated, day8, rep4

city: Tucson

first: Felicia

middle: Dare

last: Goodrum

state: AZ

address: 1657 E Helen St

department: Immunobiology

institute: University of Arizona

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using trimmomatic (parameters - LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36)
Trimmed sequence reads were mapped to concatenate GRCh38/CMV using STAR
Read count extraction and normalization were performed using subread
Assembly: Concatenated Homo_sapiens.GRCh38/Human herpesvirus 5 strain TB40/E clone TB40-BAC4
Supplementary files format and content: Microsoft Excel worksheet contains raw counts for each sample
Supplementary files format and content: Microsoft Excel worksheet contains RPKM values for each sample

source_name: peripheral blood

growth_protocol: THP1 monocytic cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS), βmercaptoethanol, L-glutamine and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
Total RNA was extracted with the Zymo Duet Quick-DNA/RNA Miniprep kit. Manufacturer protocols were followed for column isolation of total RNA, DNAse treatment, and RNA cleaning and concentration.
cDNA libraries were prepared with an Illumina TruSeq Stranded mRNA kit and a KAPA Dual-Indexed Adaper kit. The NGS library was quantified with a qPCR-based KAPA Library Quantification kit. Manufacturer protocols were followed.

treatment_protocol: THP1 cells were cultured non-adherently for 5 days following infection (or mock infection), then treated with TPA to stimulate differentiation or with DMSO as a solvent control. Treatment was washed from cells after 24 hours.

tag: agent

value: peripheral blood

value: monocyte

tag: genotype

value: differentiated

tag: time


title: THP1, UL138stopvirus, differentiated, day5.5, rep4


title: WSB_3_ORSAM11439_GT20-13836_heart


title: THP1, UL138stopvirus, undifferentiated, day6, rep4

value: undifferentiated


title: THP1, UL138stopvirus, undifferentiated, day8, rep4


title: Bari-IFN_2_24h

description:
Bulk RNAseq
Sample8
bulkRNAseq_CountsGt0_voom_filtered_idmap.txt


title: D4LPS [17_S17]

value: LPS


title: PWK_4_ORSAM11433_GT20-13788_heart


title: Bari-IFN_1_24h

description:
Bulk RNAseq
Sample7
bulkRNAseq_CountsGt0_voom_filtered_idmap.txt


title: Asi_Bel1_D2

description:
Asi_bel.bw
Asi_Bel_N_peaks.narrowPeak

value: Brain


title: PWK_3_ORSAM11432_GT20-13831_heart


title: D4CON [16_S16]

value: control


title: THP1, UL138stopvirus, undifferentiated, day5, rep4


title: IFN_2_24h

description:
Bulk RNAseq
Sample6
bulkRNAseq_CountsGt0_voom_filtered_idmap.txt


title: D3H5 [15_S15]


title: THP1, UL138stopvirus, differentiated, day6, rep4


title: PWK_2_ORSAM11431_GT20-13822_heart

value: heart


title: Bari_2_24h

description:
Bulk RNAseq
Sample4
bulkRNAseq_CountsGt0_voom_filtered_idmap.txt


title: D3H10 [13_S13]


title: NZO_8_ORSAM11429_GT20-13795_heart

value: 45percent fat


title: THP1, UL138stopvirus, undifferentiated, day3, rep4


title: THP1, UL138stopvirus, undifferentiated, day1, rep4


title: VC, scRNAseq

description:
10X Genomics
Sample9

value: Vector Control, DMSO


title: IFN_1_24h

description:
Bulk RNAseq
Sample5
bulkRNAseq_CountsGt0_voom_filtered_idmap.txt

value: Kidney organoid


title: VC_2_24h

description:
Bulk RNAseq
Sample2
bulkRNAseq_CountsGt0_voom_filtered_idmap.txt


title: NZO_7_ORSAM11428_GT20-13791_heart


title: D2H5 [10_S10]


title: PWK_1_ORSAM11430_GT20-13775_heart


title: Bari_1_24h

description:
Bulk RNAseq
Sample3
bulkRNAseq_CountsGt0_voom_filtered_idmap.txt


title: NZO_6_ORSAM11427_GT20-13794_heart


title: THP1, UL135stopvirus, undifferentiated, day8, rep4


title: D3LPS [12_S12]


title: THP1, UL135stopvirus, differentiated, day6, rep4


title: D3CON [11_S11]


title: THP1, UL135stopvirus, undifferentiated, day6, rep4


title: C004-r1_10Kh_S4

city: Milan

first: Chiara

last: Lanzuolo

address: Via Francesco Sforza 35

country: Italy

institute: CNR and Istituto Nazionale Genetica Molecolare

data_processing:
Trimming. High - throughput s equencing reads were trimmed using Trimmomatic (v0.39) (Bolger, Lohse, and Usadel 2014)  using the following parameters: 2 for seed_mismatch, 30 for palindrome_thresh old, 10 for simple_threshold, 3 for leading, 3 for trailing and 4:15 for sliding window. The sequence minimum length threshold of 35 was applied to all data sets . We used the Trimmomatic “TruSeq3 - SE.fa” (for single end) and “TruSeq3 - PE - 2.fa” (for paired end) as clip files .
Alignment. After trimm ing, reads were aligned using BWA (v0.7.17 - r1188) (Li and Durbin 2009) setting – k parameter as 2 and using as reference genome the UCSC hg38 genome (only canonical chromosomes were used ) and the output saved in BAM file format .
Filtering. The PCR duplicates were marked with Picard (v2.22; https://github.com/broadinstitute/picard) MarkDupuplicates option , then filtered using Samtools (v1.9) (Li et al. 2009) . I n addition, we filtered all the reads with mapping quality lower than 1. Each sequencing lane was analyzed separately and then merged at the end of the process. For C002 - rep1 and C004 - rep2 two sequencing runs from the same library were produced and merged after alignment and filtering.
Genomic tracks. To compute reads distribution profile (genomic tracks) we used Deeptools (v3.4.3) (Ramirez et al. 2016) bam Coverage function. For these analyses the genome was binned at 50bp, the reads extended up to 250 bp and RPKM normalization method was used. We considered a genome size of 2701495761 bp (value suggested in the Deeptools manual https://deeptools.readthedocs .io/en/latest/content/feature/effectiveGenomeSize.html) and we excluded regions known to be problematics in term of sequencing reads coverage using the black list f rom the ENCODE portal ( https://www.encodeproject.org/files/ENCFF356LFX/ )
Comparisons. for relative comparisons (relative enrichment , i.e. log2 normalized ratio) between SAMMY - seq fraction s we us ed the SPP R package (v1.16.0) (Kharchenk o, Tolstorukov, and Park 2008) and R statistical environment (v3.5.2). The reads were imported from the BAM files using the “read.bam.tags” function, then filtered using “remove.local.tag.anomalies” and finally the comparison s performed using the function “get.smoothed.enrichment.mle” setting “tag.shift = 0” and “background.density.scaling = TRUE”. The resulting enrichment signal correspond s to a log2 normalized ratio between the pair of sequencing samples.
Assembly: hg38
Supplementary files format and content: Bigwiggle files (.bw) are created using Deeptools for genomic track data and with wigToBigWig (v4) starting from wiggle files generated by SPP for what concern comparisons.

source_name: skin fibroblast

growth_protocol: Cells were cultured at 37 °C, 5% CO2in DMEM high glucose supplemented with GlutaMAX(Gibco,10566-016), with further addition of 15% (v/v) FBS (Gibco, 10270106), 100 U/mL penicillin G and 100 μg/mLStreptomycin Sulfate.

extract_protocol:
For 4f-SAMMY-seq, 3 million fibroblasts were detached from the culture plate by 3 min incubation in trypsin-EDTA at 37°C, 5% CO2. After two washes in cold PBS, they were resuspended in 600 μL ofCSK-Triton buffer (10 mM PIPES pH 6.8, 100 mM NaCl, 1 mM EGTA, 300 mM Sucrose, 3 mM MgCl2, 1mM PMSF, 1 mM DTT, 0.5% Triton X-100, with protease inhibitors). After 10 min incubation on a wheel at 4°C, soluble proteins and the cytoskeletal structure were separated from the nuclei by centrifugation at 900g for 3 min at 4°C; the supernatant was labeled as S1 fraction. The pellet was then washed with an additional volume of CSK-Triton buffer, resuspended in 100 μL of CSK buffer (10 mM PIPES pH 6.8, 100 mMNaCl, 1 mM EGTA, 300 mM Sucrose, 3 mM MgCl2, 1mM PMSF, with protease inhibitors) and incubated for 60 min at 37°C with 25 U of RNase–free DNase I (Invitrogen, AM2222). To stop DNA digestion, ammonium sulphate was added in the CSK buffer to a final concentration of 250 mM. After 5 min incubation on ice, the sample was pelleted at 900g for 3 min at 4°C; the supernatant, containing digested chromatin fragments, waslabeled as S2 fraction. Afterwards, the pellet was washed with 200 μL of CSK buffer, resuspended in 100 μL of CSK-NaCl buffer (CSK buffer with NaCl final concentration increased to 2 M) and then incubated 10 min on a wheel at 4°C. At the end of the incubation, the sample was centrifuged at 2300g for 3 min at 4°C and the supernatant was labeled as S3 fraction. Finally, after two washes in 200 μL of CSK-NaCl buffer, the pellet was solubilized in 100 μL of 8M urea; the final suspension was labeled as S4 fraction. For 10kh-SAMMY-seq, 10 thousand fibroblasts were treated following 4f-SAMMY-seq protocol, halving the volume in each step and using 12.5U of DNAse I (Invitrogen, AM2222); in this way we achieved a 150 fold increase in the ratio of enzyme units per starting number of cells, while maintaining the DNase concentration unaltered. Fractions S2, S3 and S4 were diluted in TE (10 mM TrisHCl pH 7.5, 1mM EDTA pH 8.0) to a final volume of 200 μl and then incubated 90 min at 37°C with 6 μL of RNAse cocktail (Ambion, AM2286), followed by 150 min at 55° with Proteinase K (Invitrogen, AM 2548) to a final concentration of 0.2 μg/μL. Next, DNA was isolated through phenol:chloroform:isoamyl alcohol (Sigma, 77617), precipitated and resuspended in nuclease-free water. On the next day, S2 from 4f-SAMMY-seq was additionally purified using PCR DNA Purification Kit (Qiagen, 28106) and DNA fragments in this fraction were separated using AMPure XP paramagnetic beads (Beckman Coulter, A63880) to obtain S2S (< 300 bp) and S2L (> 300 bp) fractions. Beads were added to the S2 fraction in a 0.95x (v/v) ratio, so to bind fragments larger than 300bp. Magnetic separation of beads from supernatant allowed the physical separation of larger fragments (on the beads) from shorter ones (in the supernatant). Larger fragments bound on beads were then washed in 85% ethanol, resuspended in water and magnetically separated from the beads (S2L fraction). Shorter fragments in the supernatant of the first step were bound to beads by adding a further 0.85x (v/v) beads ratio to the suspension; after washing in 85% ethanol and resuspension in water, they were also detached from beads (S2S fraction). Afterwards, S2 (from 10kh-SAMMY-seq), S2L (from 4f-SAMMY-seq), S3 and S4 (from both 10kh- and 4f-SAMMY-seq) fractions were transferred to screw cap microTUBEs (Covaris, 004078) and sonicated in a Covaris M220 focused-ultrasonicator to obtain a smear of DNA fragments peaking at 200 bp (settings: water bath 20°C, peak power 30.0, duty factor 20.0, cycles/burst 50; duration: 125 sec for S2 and S2L, 175 sec for S3 and S4). Fractions were quantified using Qubit dsDNA HS Assay Kit (Invitrogen, Q32854 ) and a Qubit 4.0 fluorometer; quality control was performed by running them on an Agilent 2100 Bioanalyzer System using the High Sensitivity DNA Kit (Agilent, 5067 – 4626).
Libraries were then created from each fraction using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) and the Unique Dual Index NEBNext Multiplex Oligos for Illumina (NEB, E6440S).

treatment_protocol: No treatment has been performed on these cells.

value: patient derived skin fibroblast

tag: passage

tag: fraction

tag: protocol

tag: sample


title: D2LPS [7_S7]


title: NZO_4_ORSAM11425_GT20-13832_heart


title: D2H10 [8_S8]


title: C004-r1_10Kh_S3


title: THP1, UL138stopvirus, undifferentiated, day5.5, rep4


title: VC_1_24h

description:
Bulk RNAseq
Sample1
bulkRNAseq_CountsGt0_voom_filtered_idmap.txt


title: NZO_5_ORSAM11426_GT20-13796_heart


title: THP1, UL135stopvirus, differentiated, day5.5, rep4


title: THP1, UL135stopvirus, differentiated, day8, rep4


title: NZO_1_ORSAM11422_GT20-13799_heart


title: D1LPS [2_S2]


title: D1H10 [3_S3]


title: NZO_2_ORSAM11423_GT20-13779_heart


title: C004-r2_4f_S4 (paired-end)


title: THP1, UL135stopvirus, undifferentiated, day5, rep4


title: D1CON [1_S1]


title: C004-r1_10Kh_S2L


title: D2CON [6_S6]


title: C004-r2_4f_S2S (paired-end)


title: THP1, UL135stopvirus, undifferentiated, day1, rep4


title: C004-r2_4f_S3 (paired-end)


title: THP1, WTvirus, undifferentiated, day8, rep4


title: C004-r2_4f_S2L (paired-end)


title: C004-r1_10Kh_S2S


title: THP1, UL135stopvirus, undifferentiated, day3, rep4


title: NOD_5_ORSAM11418_GT20-13777_heart


title: THP1, WTvirus, differentiated, day8, rep4


title: NOD_6_ORSAM11419_GT20-13810_heart


title: a-Am 1_16h

city: Birmingham

first: Marco

last: Saponaro

address: Vincent Drive

department: Institute of Cancer and Genomic Sciences

country: United Kingdom

institute: University of Birmingham

description:
single timepoint analysis
BrdU-Seq quant.txt

data_processing:
Library strategy: BrdU-Seq
Paired-end BrdU-seq reads were aligned to the hg38 genome assembly using Bowtie 2 v.2.3.3.1 (Langmead et al., 2012).
BAM files were sorted and indexed using SAMtools v.1.4 (Li et al., 2009).
The reads that mapped to region DAC blacklisted for mappability by the ENCODE project were removed.
Coverage calcualtion was done using the function “quantify” in EaSeq, from the start to the end of the gene of all annotated genes
There are no processed data files for the samples "ATMi 1_16h" "ATMi 2_16h" "ATRi 1_16h" "ATRi 2_16h", as we have used these files only for metagene profiles over specific features
Genome_build: hg38
Supplementary_files_format_and_content: .txt, containing the quantifications over each gene as above described

source_name: fibroblast cells

growth_protocol: Human immortalized fibroblasts (BJ-hTERT cells) were cultured in DMEM (Sigma-Aldrich) supplemented with 10 % FBS, 2 mM L-glutamine and penicillin/streptomycin in 5 % CO2 at 37°C.

extract_protocol:
PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific)
Illumina's Nextera XT DNA Library Preparation Kit following manufacturer’s protocol
paired end on 4 Lanes (L1-L4)

treatment_protocol: Cells fully confluent were seeded at 20 % confluence in 150 mm dishes, and grown overnight. Cells were synchronized in the G0/G1 phase in serum starvation medium (DMEM supplemented with 0.2 % FBS, 2mM L-glutamine and penicillin/streptomycin) in 5 % CO2 at 37°C for 26 h, and released into S-phase with regular DMEM for the indicated timepoints. 14h after release cells were treated for 2h with ATM (10 microM) or  ATRi (4 microM), while pulsed with BrdU for the last hour. In the case of DMSO,  DRB (100 microM) or alpha-amanitin (a-Am, 3 microg/ml), cells were treated 15 h after release for 1 hour.

value: hTERT-immortalized fibroblasts

tag: skin

value: foreskin

value: a-Am

tag: brdu antibody

value: Mouse monoclonal anti-BrdU, Sigma-Aldrich, Cat#B8442


title: THP1, WTvirus, differentiated, day6, rep4


title: a-Am 2_16h

value: Mouse monoclonal anti-BrdU, Sigma-Aldrich, Cat#B8443


title: C004-r1_4f_S4


title: NZO_3_ORSAM11424_GT20-13823_heart


title: THP1, UL135stopvirus, undifferentiated, day5.5, rep4

value: peripheral blood


title: D1H5 [5_S5]

value: human monocyte-derived macrophages (hMDMs)

value: Heme (lower concertration)


title: NOD_8_ORSAM11421_GT20-13793_heart


title: NOD_4_ORSAM11417_GT20-13833_heart


title: NOD_7_ORSAM11420_GT20-13809_heart


title: DRB 2_16h

value: DRB

value: Mouse monoclonal anti-BrdU, Sigma-Aldrich, Cat#B8441


title: THP1, WTvirus, undifferentiated, day6, rep4

value: monocyte

value: undifferentiated


title: C004-r1_4f_S3


title: C004-r1_4f_S2S


title: NOD_3_ORSAM11416_GT20-13776_heart

value: heart


title: THP1, WTvirus, differentiated, day5.5, rep4

value: differentiated


title: DMSO 1_16h

value: DMSO

value: Mouse monoclonal anti-BrdU, Sigma-Aldrich, Cat#B8438


title: DMSO 2_16h

value: Mouse monoclonal anti-BrdU, Sigma-Aldrich, Cat#B8439


title: NOD_1_ORSAM11414_GT20-13818_heart

value: 45percent fat


title: THP1, WTvirus, undifferentiated, day5.5, rep4


title: ATRi 1_16h

description: single timepoint analysis

value: hTERT-immortalized fibroblasts

value: foreskin

value: ATRi

value: Mouse monoclonal anti-BrdU, Sigma-Aldrich, Cat#B8436


title: P_8M_5

city: Orth and der Donau

first: Carolin Isabel

last: Kahlig

address: Uferstrasse 15

country: Austria

institute: Takeda

data_processing:
adapter contaminations, poly-A read-throughs and low-quanlity tails were removed using bbduk (v 38.59)
reads were mapped to GRCh38 genome using STAR aligner (v.2.7)
Count matrices were generated using HTSeq-count (v 0.12)

extract_protocol:
total RNA was harvested using Qiagen miRneasy mini plus kit
Library preparation was performed with Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Austria) using 500 ng of total RNA input

value: Embryonic Kindey Cells

value: mock


title: Cast_7_ORSAM11412_GT20-13789_heart


title: leaf_h2az+AtHTA11_3

city: Atlanta

first: Roger

middle: B

last: Deal

state: GA

address: 1510 Clifton Rd NE

department: Biology

institute: Emory University

data_processing:
Reads were trimmed of adapter content using Trimgalore. Trimmed reads were mapped to the Arabidopsis thaliana TAIR10 genome assembly, converted to the BAM file format, and sorted by coordinate using STAR. Aligned reads were indexed and quality filtered using Samtools. Mapped fragments overlapping with exons were counted using featureCounts. Gene counts were then analyzed for differential enrichment using DESeq.
Assembly: TAIR10
Supplementary files format and content: tab delimited text file including raw read counts at each TAIR10 gene via featureCounts

organism: Arabidopsis thaliana

source_name: third or fourth pair of rosette leaves

growth_protocol: Plants were grown in soil in growth chambers at +20°C under a 16 hour-light/8-hour dark cycle.

extract_protocol:
Total RNA was extracted per the RNeasy Plant Mini kit (Qiagen). For h2a.z plants, above ground tissue from three different plants was harvested and individually processed to extract total RNA following the manufacturer’s recommendations. For all others, an RLT suspension of frozen leaf powder was split into three equal aliquots of 400 μl for 3 replicates. Total RNA was DNase-treated (Ambion) and quantified using Nanodrop One (Thermo Scientific). 100 nanograms for each of the three replicates from every sample were then used as input to generate RNA-seq libraries following the Universal RNA-seq with NuQuant protocol (Tecan).
RNA-seq libraries were constructed following the Universal RNA-seq with NuQuant protocol (Tecan).

value: third or fourth pair of rosette leaves


title: NOD_2_ORSAM11415_GT20-13792_heart


title: Cast_8_ORSAM11413_GT20-13821_heart


title: THP1, WTvirus, undifferentiated, day3, rep4

value: peripheral blood


title: THP1, WTvirus, undifferentiated, day1, rep4


title: ATMi 2_16h

description: single timepoint analysis

value: ATMi

value: Mouse monoclonal anti-BrdU, Sigma-Aldrich, Cat#B8435


title: Cast_6_ORSAM11411_GT20-13805_heart


title: THP1, uninfected, differentiated, day8, rep4

value: monocyte


title: DRB 1_16h

value: DRB

value: Mouse monoclonal anti-BrdU, Sigma-Aldrich, Cat#B8440


title: P_8M_13


title: Cast_5_ORSAM11410_GT20-13800_heart

value: heart


title: Control_DMSO2_D1

description:
Control_DMSO.bw
Control_DMSO_N_peaks.narrowPeak

value: Brain

value: Glioblastoma


title: C004-r1_4f_S2L

value: patient derived skin fibroblast


title: ATRi 2_16h

description: single timepoint analysis

value: hTERT-immortalized fibroblasts

value: Mouse monoclonal anti-BrdU, Sigma-Aldrich, Cat#B8437


title: P_8M_1


title: THP1, uninfected, undifferentiated, day8, rep4

value: undifferentiated


title: THP1, uninfected, differentiated, day6, rep4

value: differentiated


title: P_8M_17

value: Embryonic Kindey Cells


title: P_8M_9

value: mock


title: Cast_3_ORSAM11409_GT20-13824_heart

value: 45percent fat


title: THP1, WTvirus, undifferentiated, day5, rep4

value: peripheral blood


title: a-Am 1 22h

description:
expression analysis at the specific timepoint
aam_ctr_22_only

data_processing:
Single-end RNA-seq data were aligned to the hg38 genome assembly using STAR v.020201
Reads were trimmed to remove RNA contamination via BBDuk
BAM files were sorted and indexed using SAMtools
Counts for each transcribed gene were computed by featureCounts using the annotation of the GENCODE genes
Differential expression analysis in RNA-seq data was conducted by using the package DESeq2
Genome_build: hg38
Supplementary_files_format_and_content: .txt, containing the quantifications over each gene as above described

extract_protocol:
RNA extracted with RNeasy MiniKit (QIAGEN)
RNA libraries were prepared according to NEBNext Ultra II mRNA Kit (NEB)
single end on 4 Lanes (L1-L4)

treatment_protocol: Cells fully confluent were seeded at 20 % confluence in 150 mm dishes, and grown overnight. Cells were synchronized in the G0/G1 phase in serum starvation medium (DMEM supplemented with 0.2 % FBS, 2mM L-glutamine and penicillin/streptomycin) in 5 % CO2 at 37°C for 26 h, and released into S-phase with regular DMEM for the indicated timepoints. 15h or 21h  after release cells were treated for 1h with DMSO,  DRB (100 microM) or alpha-amanitin (a-Am, 3 microg/ml), followed by RNA extraction.

value: foreskin

value: a-Am

tag: time point


title: a-Am 2 22h


title: Cast_1_ORSAM11407_GT20-13780_heart


title: a-Am 3 22h


title: Cast_2_ORSAM11408_GT20-13835_heart


title: THP1, uninfected, differentiated, day5.5, rep4

value: monocyte


title: DRB 3 22h

description:
expression analysis at the specific timepoint
DRB_ctr_22_only

value: DRB


title: P_7M_5


title: B6_8_ORSAM11406_GT20-13802_heart


title: P_7M_9


title: THP1, uninfected, undifferentiated, day5.5, rep4


title: P_7M_17


title: P_7M_13


title: B6_7_ORSAM11405_GT20-13820_heart


title: DRB 2 22h


title: P_7M_1


title: THP1, uninfected, undifferentiated, day5, rep4


title: B6_6_ORSAM11404_GT20-13803_heart


title: P_6M_9


title: THP1, uninfected, undifferentiated, day3, rep4


title: DMSO 3 22h

description:
expression analysis at the specific timepoint
DRB_ctr_22_only, aam_ctr_22_only

value: DMSO


title: DRB 1 22h


title: B6_5_ORSAM11403_GT20-13808_heart

value: heart


title: ATMi 1_16h

description: single timepoint analysis

value: ATMi

value: Mouse monoclonal anti-BrdU, Sigma-Aldrich, Cat#B8434


title: DMSO 2 22h


title: P_6M_5


title: THP1, UL138stopvirus, differentiated, day8, rep3


title: P_6M_17


title: B6_4_ORSAM11402_GT20-13829_heart


title: a-Am 3 16h

description:
expression analysis at the specific timepoint
aam_ctr_16_only


title: DMSO 1 22h


title: THP1, UL138stopvirus, differentiated, day6, rep3


title: THP1, UL138stopvirus, undifferentiated, day6, rep3


title: P_6M_1


title: P_6M_13


title: B6_1_ORSAM11399_GT20-13826_heart


title: a-Am 2 16h


title: B6_2_ORSAM11400_GT20-13812_heart


title: AJ_8_ORSAM11398_GT20-13816_heart


title: a-Am 1 16h


title: THP1, UL138stopvirus, differentiated, day5.5, rep3


title: P_5M_9


title: THP1, uninfected, undifferentiated, day1, rep4


title: THP1, UL138stopvirus, undifferentiated, day5.5, rep3


title: P_5M_5


title: AJ_6_ORSAM11397_GT20-13786_heart


title: P_5M_17


title: P_5M_13


title: AJ_5_ORSAM11396_GT20-13814_heart


title: B6_3_ORSAM11401_GT20-13828_heart


title: DRB 2 16h

description:
expression analysis at the specific timepoint
DRB_ctr_16_only


title: DRB 1 16h


title: THP1, UL138stopvirus, undifferentiated, day3, rep3


title: AJ_4_ORSAM11395_GT20-13801_heart


title: DMSO 3 16h

description:
expression analysis at the specific timepoint
DRB_ctr_16_only, aam_ctr_16_only


title: DMSO 2 16h


title: P_4T_9

value: transfected


title: THP1, UL135stopvirus, differentiated, day8, rep3


title: AJ_3_ORSAM11394_GT20-13782_heart


title: P_4T_5


title: AJ_2_ORSAM11393_GT20-13798_heart


title: DMSO 1 16h

value: hTERT-immortalized fibroblasts


title: AJ_1_ORSAM11392_GT20-13790_heart


title: P_4T_17


title: PCNA 16h ChIP-Seq

data_processing:
Single-end ChIP-seq reads were aligned to the hg38 genome assembly using Bowtie 2 v.2.3.3.1 (Langmead et al., 2012).
BAM files were sorted and indexed using SAMtools v.1.4 (Li et al., 2009).
The reads that mapped to region DAC blacklisted for mappability by the ENCODE project were removed.
Peaks were called in BrdU samples (from GSE136288) against the relative Input DNA using MACS2 v.2.1.0 with human genome size and the following parameters: -m 8 30, -p 0.00001 in two repeats. BrdU peaks identified in each timepoint were all collected together in the file all_peals.txt
Metagenes were generated using the function “average” in EaSeq at TSS, BrdU peaks and other genomic features
Assembly: hg38
Supplementary files format and content: .txt contains the list of all the BrdU peaks identified

source_name: skin; foreskin

extract_protocol:
Cells were fixed in 1 % formaldehyde for 10 min at room temperature. Then, glycine was added to a final concentration of 0.125 M and the reaction was incubated for 5 min at room temperature. Fixed cells were washed twice with ice-cold PBS, and lysed for 5 min in ice-cold ChIP cell lysis buffer (5 mM HEPES pH 8.0, 85 mM KCl, 0.5 % NP-40 alternative, protease inhibitors). Nuclei were pelleted by centrifugation at 3900 g for 5 min at 4ºC, and lysed for 5 min in ChIP nuclear lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1 % SDS, protease inhibitors) and incubated 5 minutes on ice. Lysates were sonicated using VCX130 (Sonics & Materials, Inc) with the following settings: 8 cycles, 30 % Amp, 15 s ON, 25 s OFF. Sonicated chromatin was centrifuged at 17000 g at 4ºC for 10 min and the supernatant was stored at -80ºC until immunoprecipitation. Before immunoprecipitation, 15 µl of Dynabeads Protein A was washed twice with 900 µl 5 mg/ml BSA in PBS and reacted for 1 h or more with 3 µg of the corresponding antibody (Phospho-Histone H2A.X: ab2893, Abcam; H3: ab1791, Abcam) in 500 µl of 5 mg/ml BSA in PBS at room temperature. The beads were washed again with 900 µl 5 mg/ml BSA in PBS before use. The concentration of sonicated chromatin was quantified by using Quick Start Bradford protein 1x dye reagent (Bio-Rad). 50 µl chromatin were used as Input. 0.6 mg of chromatin was 1:5 diluted with ChIP dilution buffer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, protease inhibitors), and incubated with prepared beads overnight at 4ºC with rotation. Chromatin bound to beads was washed twice in ChIP low salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), twice in ChIP high salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl),  twice in ChIP LiCl buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 % NP40 alternative, 1 % sodium deoxycholate, 1 mM EDTA), and once in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Most of the TE buffer was removed after the beads were centrifuged at 3000 rpm for 3 min and put on Magnet. The remaining TE was removed after the beads were centrifuged again at 13200 rpm for 1 min and put on Magnet. The beads were resuspended in 50 µl elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1 % SDS) and incubated at 65°C for 10 min followed by centrifugation at 12000 rpm for 1 min. The supernatant was transferred to a new DNA low binding tube (Sarstedt) and incubated at 65°C overnight for de-crosslinking. Reverse cross-linked supernatant was mixed with 50 µl of TE and incubated with RNase A at 37°C for 1 h. The supernatant was further incubated with 0.7 mg/ml proteinase K at 55°C for 2 h. DNA was purified by DNA Clean & Concentrator-5 and eluted with 8 µl EB (10 mM Tris-HCl pH 8.5). The purified DNA was quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). 1 ng of DNA was used to construct DNA library according to Illumina's Nextera XT DNA Library Preparation Kit protocol. After being cleaned up, indexed libraries were quantified, normalized and pooled, sequenced on Illumina NextSeq system
NEBNext® Ultra™ II DNA Library Prep with Sample Purification Beads following manufacturer’s protocol
single end on 4 lanes (L1-L4)

value: skin; foreskin

value: BJ hTERT

value: fibroblasts

tag: chip antibody

tag: chip antibody_vendor

value: The Francis Crick Institute

tag: chip antibody_cat._#


title: THP1, UL135stopvirus, undifferentiated, day8, rep3

tag: tissue

tag: cell type

value: undifferentiated


title: DRB 3 16h


title: THP1, UL138stopvirus, undifferentiated, day5, rep3

tag: cell line

tag: treatment


title: THP1, UL135stopvirus, differentiated, day6, rep3

tag: agent

value: differentiated


title: 129_9_ORSAM11391_GT20-13787_heart

tag: diet

value: 45percent fat


title: P_4T_13

value: Embryonic Kindey Cells

tag: genotype

tag: time


title: 129_8_ORSAM11390_GT20-13813_heart


title: THP1, UL135stopvirus, differentiated, day5.5, rep3

value: peripheral blood


title: THP1, UL138stopvirus, undifferentiated, day1, rep3

value: monocyte


title: P_4T_1


title: PCNA 14h ChIP-Seq


title: RNAPII 16h ChIP-Seq

value: Rpb1 NTD

value: Cell Signaling Technology


title: P_5M_1

value: mock


title: THP1, UL135stopvirus, undifferentiated, day5.5, rep3


title: RNAPII 14h ChIP-Seq


title: P_3T_9


title: P_3T_5


title: THP1, UL135stopvirus, undifferentiated, day5, rep3


title: P_3T_17


title: 129_4_ORSAM11387_GT20-13830_heart

value: heart


title: 129_3_ORSAM11386_GT20-13785_heart


title: MCF7 Triptolide Vehicle Rep2

city: Edinburgh

first: Luciana

middle: Ines

last: Gomez Acuna

address: Crewe Road

institute: MRC-University of Edinburgh

data_processing:
Capture-C data were aligned to the hg19 genome and processed using distiller-nf 0.3.3 (https://doi.org/10.5281/zenodo.3350937) with default settings.
Coolers filtered for q>=30 were used and ICE balanced (Imakaev et al., 2012) using the cooler (Abdennur et al., 2020) command 'cooler balance --cis_only --blacklist blacklist.bed' where blacklist.bed contains all regions outside of the capture probe area.
Assembly: hg 19
Supplementary files format and content: Cooler files
Library strategy: Capture-C-seq

growth_protocol: MCF7 cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS) and antibiotics in humidified atmosphere with 5% CO2 at 37C. Before treatment, cells were grown in phenol red-free DMEM supplemented with 10% charcoal stripped FCS for 4 days.

extract_protocol:
Cells were fixed with 1 .5 % formaldehide for 10 minutes. After lisis, nuclei were subjeted to in situ DpnII restriction and ligation. DNA was decrosslinked and extracted with phenol:chloroform. Regions of interest were captured from 3C libraries using fragmented and biotinilated tiled BACs. Captured regions' coordenates (hg19): chr2:11450601-11945472; chr21:16143548-17096678
Before Capture, 3C libraries were prepaired with NEBNext® Ultra™ II DNA Library Prep for Illumina

treatment_protocol:
Cells were treated with either vehicle (ethanol) or E2 10nM for 5, 30 or 60 minutes.
WT, GREB1 enhacner deletion or NRIP1 enhancer deletion were treated with either vehicle (ethanol) or E2 10nM for 30 minutes.
Cells were treated with either vehicle (ethanol), Tamoxifen 10 nM or  E2 10nM 30 minutes.
Cells were treated with either vehicle (DMSO), Flavopiridol 10 μM or Triptolide 10 μ for 5 min and then, with either vehicle (ethanol) or E2 10nM for additional 30 minutes.

value: Mammary gland, epithelial

value: WT

value: Triptolide 10μ 5 min, Vehicle 30 min


title: P_3T_13

value: transfected


title: THP1, UL135stopvirus, undifferentiated, day1, rep3


title: 129_2_ORSAM11385_GT20-13817_heart


title: MCF7 Flavopiridol E2 Rep2

value: Flavopiridol 10μ 5 min, E2 10 nM 30 min


title: THP1, WTvirus, differentiated, day8, rep3


title: P_3T_1


title: MCF7 Flavopiridol Vehicle Rep2

value: Flavopiridol 10μ 5 min, Vehicle 30 min


title: 129_7_ORSAM11389_GT20-13827_heart


title: THP1, UL135stopvirus, undifferentiated, day6, rep3


title: THP1, WTvirus, undifferentiated, day8, rep3


title: MCF7 DMSO E2 Rep2

value: DMSO 5 min, E2 10 nM 30 min


title: 129_1_ORSAM11384_GT20-13804_heart


title: THP1, WTvirus, differentiated, day6, rep3


title: P_2T_5


title: microglia,SPF_Sham,Day7_3

city: hangzhou

first: Xuchao

last: He

state: zhejiang

address: 88 jiefang RD

institute: zhejiang university

data_processing:
Adaptor contamination was removed using Cutadapt 1.9 software. HISAT2 2.0.4 software mapped the reads to the genome, and StringTie 1.3.4d assembled the mapped reads of each sample. Expression levels were estimated and calculated as TPM (TPM = (exon reads in gene) / (total exon reads) × 1 million). Differentially expressed mRNAs were identified using edgeR, selecting those with a fold change > 2 or fold change < 0.5 and a p-value < 0.05, and annotated with GO terms and KEGG Pathways.
Assembly: mm10
Supplementary files format and content: tab-delimited text files includes raw counts for each counts

source_name: brain

extract_protocol:
Sorted microglia from peri-hematoma were collected in 2mL TRIzol reagent and stored at -80℃.
Gene expression at the mRNA level was analyzed using the SMART-Seq 2 protocol. Specifically, the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech, Japan) was employed to prepare the low-input library as follows: 1) Total RNA of pg level was used to prepare the cDNA library; 2) cDNA was purified and size-selected within the 150-300 bp range; 3) A sequencing-ready library was prepared by adding i5/i7 adaptors using the PCR method; 4) Library quality was assessed using an Agilent 2100 Bioanalyzer to determine size and concentration; 5) Libraries were sequenced on an Illumina Novaseq™ 6000 platform using a 2 × 150 bp paired-end sequencing protocol.

value: brain

value: microglia

value: none,PBS


title: MCF7 DMSO Vehicle Rep2

value: DMSO 5 min, Vehicle 30 min


title: P_2T_9

value: Embryonic Kindey Cells


title: microglia,SPF_Sham,Day7_2


title: MCF7 Triptolide E2 Rep2

value: Triptolide 10μ 5 min, E2 10 nM 30 min


title: 129_6_ORSAM11388_GT20-13819_heart

value: 45percent fat


title: P_2T_17


title: THP1, WTvirus, undifferentiated, day6, rep3

value: peripheral blood

value: monocyte

value: undifferentiated


title: THP1, UL135stopvirus, undifferentiated, day3, rep3


title: microglia,SPF_Sham,Day7_1


title: MCF7 Triptolide E2 Rep1

value: Mammary gland, epithelial


title: THP1, WTvirus, differentiated, day5.5, rep3

value: differentiated


title: P_2T_13


title: MCF7 Triptolide Vehicle Rep1

value: WT

value: Triptolide 10μ 5 min, Vehicle 30 min


title: microglia,SPF_ICH,Day7_3

value: none,collagenase


title: P_2T_1

value: transfected


title: microglia,SPF_ICH,Day7_2


title: THP1, WTvirus, undifferentiated, day5, rep3


title: MCF7 Flavopiridol E2 Rep1

value: Flavopiridol 10μ 5 min, E2 10 nM 30 min


title: MCF7 Flavopiridol Vehicle Rep1

value: Flavopiridol 10μ 5 min, Vehicle 30 min


title: P_1T_9


title: THP1, WTvirus, undifferentiated, day5.5, rep3


title: THP1, WTvirus, undifferentiated, day3, rep3


title: P_1T_5


title: MCF7 DMSO E2 Rep1

value: DMSO 5 min, E2 10 nM 30 min


title: microglia,SPF_ICH,Day3_3

value: brain

value: microglia


title: microglia,SPF_ICH,Day7_1


title: P_1T_17

value: Embryonic Kindey Cells


title: microglia,SPF_ICH,Day3_2


title: P_1T_13


title: MCF7 DMSO Vehicle Rep1

value: DMSO 5 min, Vehicle 30 min


title: THP1, uninfected, undifferentiated, day8, rep3

value: mock


title: microglia,SPF_ICH,Day3_1


title: THP1, uninfected, differentiated, day8, rep3

value: peripheral blood


title: MCF7 NRIP1enhDel clone2 E2 Rep2

value: NRIP1 enhancer deletion

value: E2 10 nM 30 min


title: microglia,GF_Sham,Day7_3

value: germ free condition,PBS


title: P_1T_1


title: microglia,GF_Sham,Day7_2


title: I_8M_9


title: THP1, WTvirus, undifferentiated, day1, rep3

value: monocyte

value: undifferentiated


title: I_8M_5


title: MCF7 NRIP1enhDel clone2 vehicle Rep2

value: Mammary gland, epithelial

value: Vehicle


title: THP1, uninfected, undifferentiated, day6, rep3


title: THP1, uninfected, differentiated, day5.5, rep3

value: differentiated


title: microglia,GF_Sham,Day7_1

value: WT


title: THP1, uninfected, differentiated, day6, rep3


title: MCF7 GREB1enhDel clone2 E2 Rep2

value: GREB1 enhancer deletion


title: MCF7 NRIP1enhDel clone1 vehicle Rep2


title: I_8M_17


title: microglia,GF_ICH,Day7_3

value: germ free condition,collagenase


title: THP1, uninfected, undifferentiated, day5.5, rep3


title: MCF7 GREB1enhDel clone2 vehicle Rep2


title: I_8M_13


title: microglia,GF_ICH,Day7_2

value: brain


title: I_8M_1


title: THP1, uninfected, undifferentiated, day5, rep3


title: THP1, uninfected, undifferentiated, day6, rep4


title: THP1, uninfected, undifferentiated, day3, rep3


title: MCF7 GREB1enhDel clone1 vehicle Rep2


title: microglia,GF_ICH,Day7_1

value: microglia


title: MCF7 GREB1enhDel clone1 E2 Rep2


title: MCF7 NRIP1enhDel clone1 E2 Rep2


title: microglia,GF_ICH,Day3_3


title: THP1, uninfected, undifferentiated, day1, rep3


title: I_7M_9

value: Embryonic Kindey Cells


title: I_7M_5


title: MCF7 WT E2 Rep2


title: microglia,GF_ICH,Day3_2


title: THP1, UL138stopvirus, differentiated, day8, rep2


title: microglia,GF_ICH,Day3_1


title: I_7M_13


title: MCF7 NRIP1enhDel clone2 E2 Rep1


title: microglia,ABX_Sham,Day7_3

value: antibiotic cocktail,PBS


title: I_7M_17

value: mock


title: I_7M_1


title: THP1, UL138stopvirus, differentiated, day6, rep2

value: peripheral blood


title: THP1, UL138stopvirus, undifferentiated, day6, rep2


title: I_6M_9


title: microglia,ABX_Sham,Day7_2


title: MCF7 NRIP1enhDel clone1 E2 Rep1

value: NRIP1 enhancer deletion


title: microglia,ABX_Sham,Day7_1


title: MCF7 NRIP1enhDel clone2 vehicle Rep1


title: THP1, UL138stopvirus, differentiated, day5.5, rep2


title: THP1, UL138stopvirus, undifferentiated, day5.5, rep2


title: I_6M_17


title: MCF7 GREB1enhDel clone2 E2 Rep1

value: E2 10 nM 30 min


title: I_6M_5


title: THP1, UL138stopvirus, undifferentiated, day5, rep2


title: microglia,ABX_ICH,Day7_2

value: antibiotic cocktail,collagenase


title: MCF7 NRIP1enhDel clone1 vehicle Rep1


title: MCF7 WT vehicle Rep2


title: THP1, UL138stopvirus, undifferentiated, day3, rep2

value: monocyte


title: THP1, UL138stopvirus, undifferentiated, day8, rep2

value: undifferentiated


title: I_6M_1


title: microglia,ABX_ICH,Day3_3


title: MCF7 GREB1enhDel clone1 E2 Rep1

value: Mammary gland, epithelial


title: THP1, UL138stopvirus, undifferentiated, day1, rep2


title: I_5M_9


title: THP1, UL135stopvirus, differentiated, day8, rep2


title: microglia,ABX_ICH,Day3_2


title: I_5M_5


title: I_6M_13


title: MCF7 WT E2 Rep1


title: microglia,ABX_ICH,Day3_1


title: microglia,ABX_ICH,Day7_3


title: THP1, UL135stopvirus, undifferentiated, day8, rep2


title: MCF7 WT vehicle Rep1

value: Vehicle


title: microglia,ABX_ICH,Day7_1


title: MCF7 TC E2 60min Rep2

value: E2 10 nM 60 min


title: I_5M_13


title: MCF7 GREB1enhDel clone2 vehicle Rep1


title: THP1, UL135stopvirus, undifferentiated, day6, rep2


title: THP1, UL135stopvirus, differentiated, day6, rep2

value: differentiated


title: I_5M_1


title: MCF7 TC E2 30min Rep2

value: WT


title: THP1, UL135stopvirus, differentiated, day5.5, rep2


title: Bone marrow, conditioned and transplanted, GFP+ CD45+ CD11b+ cells (n1, n2, n3)

city: Stanford

first: Natalia

last: Gomez-Ospina

address: 265 Campus Drive

institute: Stanford

description: Cells were isolated from two mice and pooled for scRNA-seq

data_processing:
Raw Illumina BCL sequencing files were processed through bcl2fastq (v2.20) to generate FASTQ files   (https://support.10xgenomics.com/single-cell-gene-expression/software/overview/welcome)
FASTQ files were processed through Cellranger count  (v7.1.0) to obtain counts matrices
Assembly: mm10
Supplementary files format and content: Tab-separated value files and matrix files (for all samples)

source_name: Bone  marrow

extract_protocol:
The brain was minced in ice-cold HBSS 1X (Thermo Fisher Scientific 14185052), the pieces were collected by centrifugation at 400g for 5 minutes and digested using the NTDK Neural Tissue Dissociation Kit (P) (Miltenyi Biotec 130-092-628) at 37° C for 30 minutes following the manufacturer’s guidelines. Digested samples were quenched using ice-cold HBSS 1X and filtered through a 70 μm cell strainer, resuspended in 33% isotonic Percoll PLUS (GE Healthcare GE17-5445-01) and spun at 700 g for 15 minutes for myelin removal. The cell pellet was washed with FACS-BL buffer (PBS-1X supplemented with 5% Fetal Bovine Serum and 1% BSA fraction V) and kept on ice for FACS staining. Bone marrow cells (BM, from femurs and tibiae) were extracted in RPMI supplemented with 10% FBS, 4U/mL Heparin and 0.2U/mL Dnase I and filtered using a 30 μm cell strainer and kept on ice for FACS staining. For FACS staining, cells were resuspended in 10% vol/vol Mouse BD Fc Block™ (clone 2.4G2 BD Biosciences) for 10 minutes and stained in the dark for 30 min using the following fluorophore-tagged antibodies: anti-mouse CD45 PE-Cy7 (clone 104 Thermo Fisher Scientific), anti-mouse CD45 PE-Cy7 (clone 30F11 Thermo Fisher Scientific), anti-mouse/human CD11b-PE (clone M1/70 BioLegend), anti-mouse/human TER-119 PE-Cy5 (TER-119, BD Biosciences), anti-mouse Ly6C BV605 (clone AL-21 BD Biosciences), anti-mouse CD3 APC (clone 17A2 Thermo Fisher Scientific), anti-mouse CD19 PB (clone 6D3 BioLegend); eFluor™780 (Thermo Fisher Scientific 65-0865-18) was used as a viability dye.   Cell Hashing, using oligo-tagged antibodies directed to mouse CD45 (clone 30-F11) and mouse MHCI (clone M1/42), was used to uniquely label cells from distinct mice. The sequencing of each mouse-specific tag alongside the cellular transcriptome allowed us to pool cells from three different mice per sample and prevent sample-specific batch effects that occur during single-cell RNA sequencing. Each unique oligo-tagged antibody mix was added to the flow cytometry antibody mix described above (0.15 µL/1x106 cells). After staining, cells were washed and resuspended in FACS-BL buffer. Stained cells were sorted using a MA900 Multi-Application Cell Sorter (Sony Biotechnology) flow cytometer in cold FACS-BL buffer. The unique oligo-tagged antibodies were as follows: MGLC, host MG, BM-CD11b+ mouse 1 (TotalSeqTM-B 0301 anti-mouse Hashtag 1 Antibody, BioLegend 155831), mouse 2 (TotalSeqTM-B 0302 anti-mouse Hashtag 2 Antibody, BioLegend 155833), mouse 3 (TotalSeqTM-B 0303 anti-mouse Hashtag 3 Antibody, BioLegend 155835); Naïve MG mouse 1 (TotalSeqTM-B 0301 anti-mouse Hashtag 1 Antibody, BioLegend 155831), mouse 2 (TotalSeqTM-B 0302 anti-mouse Hashtag 2 Antibody, BioLegend 155833), mouse 3 (TotalSeqTM-B 0303 anti-mouse Hashtag 3 Antibody, BioLegend 155835).
Cells were processed using the Chromium Controller and the Chromium Next GEM Single Cell 3' Reagent Kits (10x Genomics). The libraries were sequenced via Illumina Novaseq 6000 sequencer.

value: Bone  marrow

value: CD45+ CD11b+ myeloid

tag: Sex

value: female

tag: age

value: 10.8 month-old

tag: strain

value: Busulfan + PLX3397 + Transplant of Lineage negative KIT+ SCA1+ HSPCs expanded in culture


title: I_4T_9

value: Embryonic Kindey Cells

value: transfected


title: Brain, untreated, GFP+ CD45+ CD11b+ cells (n1, n2, n3)

value: Brain

value: CD45+ CD11b+ microglia/myeloid

value: Untreated


title: MCF7 TC E2 5min Rep2

value: E2 10 nM 5 min


title: MCF7 GREB1enhDel clone1 vehicle Rep1

value: GREB1 enhancer deletion


title: Brain, conditioned and transplanted, GFP+ CD45+ CD11b+ cells (n1, n2, n3)


title: THP1, UL135stopvirus, undifferentiated, day5.5, rep2

value: peripheral blood

value: monocyte

value: undifferentiated


title: Brain, conditioned and transplanted, GFP- CD45+ CD11b+ cells (n1, n2, n3)

value: C57BL/6J (WT)

value: Busulfan + PLX3397 +  Transplant of Lineage negative KIT+ SCA1+ HSPCs expanded in culture


title: MCF7 TC Vehicle Rep2

value: Mammary gland, epithelial

value: WT

value: Vehicle


title: TDLN_DC_scATACseq_Sample_2

city: Durham

first: Brent

last: Hanks

state: North Carolina

address: 308 Research Dr

department: Medicine

institute: Duke University

description: 10x Genomics Next Gem Single Cell ATACseq

library_source: genomic single cell

data_processing:
Sequencing was demultiplexed, the barcodes were identified and the genes were counted using the CellRanger atac 2.0.0 pipeline (https://www.10xgenomics.com/support/software/cell-ranger/release-notes/cr-release-notes)
Assembly: mm10
Supplementary files format and content: tab separated values, matrix files and BED files

source_name: Tumor-draining lymph node  (inguinal)

growth_protocol: B6.Cg-Tg(Tyr-cre/ERT2)13BosBraftm1MmcmPtentm1Hwu/BosJ mice from Jackson Lab were induced with 4HT on the back of the mice .  Tumor  were allowed to for 4 weeks at which point the inguinal TDLN or axillary non-draining LN was collected.

extract_protocol:
Lymph nodes were digested in collagenase and Dnase and DCs were collected by FACS sorting for Lin- Cd11c+ MHCIIhi cells
Libraries were constructed using 10x Genomics default protocols following the Chromium 3' v3.1

value: Tumor-draining lymph node  (inguinal)

value: 4 weeks following induction

value: Dendritic Cells

value: BRafCA, PtenloxP, Tyr::CreERT2

value: 4-hydroxy-tamxifen tumor induction


title: I_5M_17

value: Embryonic Kindey Cells

value: mock


title: I_4T_17

value: transfected


title: TDLN_DC_scATACseq_Sample_1


title: THP1, UL135stopvirus, undifferentiated, day3, rep2


title: MCF7 TC E2 30min Rep1

value: E2 10 nM 30 min


title: I_4T_13


title: MCF7 TC E2 60min Rep1

value: E2 10 nM 60 min


title: Tumor_CITE

description: 10x Genomics Next Gem Single Cell 3' Gene Expression

data_processing:
Sequencing was demultiplexed, the barcodes were identified and the genes were counted using the CellRanger 6.0.1 pipeline and cell ranger multi was used to resolve cell hashtags (https://www.10xgenomics.com/support/software/cell-ranger/release-notes/cr-release-notes)
Assembly: mm10
Supplementary files format and content: tab seperated values and matrix files

source_name: Mouse Melanoma tumor

growth_protocol: B6.Cg-Tg(Tyr-cre/ERT2)13BosBraftm1MmcmPtentm1Hwu/BosJ mice from Jackson Lab were induced with 4HT on the back of the mice .  Tumor  were allowed to for 4 weeks at which point the inguinal TDLN or tumor was collected.

extract_protocol:
Lymph nodes were digested in collagenase and Dnase and tumor was digested using collagenase DNase and Hyaluronidase. DCs were collected by FACS sorting for Lin- Cd11c+ MHCIIhi cells.  During the antibody staining process CITEseq oligo tagged antibodies were incubated along with the fluorescence conjugated antibodoies for cell sorting. For cell hashing, an n=2 was used with DCs sorted from two independent mice and labeled with either Totalseq-B0301 Hashtag 1 or TotalseqB0302 Hashtag2.
Libraries were constructed using 10x Genomics default protocols following the Chromium 3' v3.1. A separate antibody CITEseq barcode library was created using the 10x Genomics feature barcoding primer set.

value: Mouse Melanoma tumor

value: 4 weeks following  4-hydroxy-tamxifen tumor induction


title: I_4T_1


title: THP1, WTvirus, differentiated, day8, rep2

value: peripheral blood

value: monocyte

value: differentiated


title: I_3T_9


title: THP1, WTvirus, undifferentiated, day8, rep2

value: undifferentiated


title: TDLN_CITE

source_name: Inguinal lymph node of tumor bearing mouse


title: THP1, UL135stopvirus, undifferentiated, day1, rep2


title: I_3T_5


title: I_4T_5


title: MCF7 TC E2 5min Rep1

value: Mammary gland, epithelial

value: WT

value: E2 10 nM 5 min


title: MCF7 TC VehicleRep1

value: Vehicle


title: THP1, WTvirus, differentiated, day6, rep2


title: MCF7 E2 10 nM 60 min Rep2

description:
LGA_4_2
There are 4 raw data files (L001-L004) per each read end (R1 and R2)

data_processing:
For each sample and read end, multiple raw fastq files were merged.
Sequence reads were trimmed for adaptor sequence/low-quality sequence and short reads were removed using Cutadapt v1.16 in paired end mode (options: -q 20 -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCA  -A AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT -m 20)
Trimmed sequence reads were mapped to hg19 using STAR v2.7.8 for paired end data and using Ensemble GRCCh37gene annotation to generate BAM files (options: --outFilterType BySJout --outFilterMultimapNmax 20 --outSAMunmapped Within). Bam files were indexed using samtools (v1.3) index.
Bigwig files used for data visualization on UCSC genome browser, one file per strand, were generated using deeptools (v3.5.1) bamCoverage (options: --normalizeUsing RPKM -bs 1 --filterRNAstrand forward or reverse).
Read counts over regions 1 kb downstream of TSSs from Ensemble GRCCh37gene annotation were calculated using featureCounts
Assembly: hg19
Supplementary files format and content: Two bigwig files per sample, one for the plus and one for the minus strand.
Supplementary files format and content: Raw read counts over TSSs regions

extract_protocol:
Toral RNA was extracted with TRIZOL (Invitrogen) and sonicated. 4sU RNA was labelled with biotin and biotinilated RNA was purified with µMacs Streptavidin Kit (Miltenyi).
RNA-seq llibraries were prepaired using theNEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®.

treatment_protocol: Cells were treated with either vehicle (ethanol) or E2 10nM for 5, 30 or 60 minutes. 5 minutes before harvesting, 4sU to a final concentration of 500 μM was incorporated to the media.


title: I_3T_17


title: Ctrl_LN_CITE

source_name: Inguinal lymph node

value: Inguinal lymph node

value: 10 week old mice

value: Dendritic Cells


title: MCF7 E2 10 nM 60 min Rep1

description:
LGA_4_1
There are 4 raw data files (L001-L004) per each read end (R1 and R2)


title: 2170_2171_Pos

city: Oklahoma City

first: Willard

last: Freeman

address: 825 NE 13th St

institute: Oklahoma Medical Research Foundation

data_processing:
Following sequencing, reads were trimmed, aligned, differential expression statistics, and correlation analyses were performed using Strand NGS software (Version 3.1, Bangalore, India)
Reads were aligned against the Mm10 build of the mouse genome with RefSeq Genes and Transcripts (2013.04.01) annotation build. Alignment and filtering criteria included: adapter trimming, fixed 2bp trim from 5’ and 6bp from 3’ ends, a maximum number of one novel splice allowed per read, a minimum of 90% identity with the reference sequence, a maximum of 5% gap, trimming of 3’ end with Q<30.
Alignment was performed directionally with Read 1 aligned in reverse and Read 2 in forward orientation. Reads were filtered based on the mapping status and only those reads that aligned normally (in the appropriate direction) were retained. Duplicate aligning reads were then removed.
Normalization was performed with the DESeq algorithm.
Transcripts with an average read count value ≥50 in at least 100% of the samples in at least one group were considered expressed at a level sufficient for quantitation and those transcripts below this level were considered not detected/not expressed and excluded, as these low levels of reads are close to background and are highly variable.
For those transcripts meeting this statistical criterion, a fold change >|1.25| cutoff was used to eliminate those genes which were statistically significant but unlikely to be biologically significant and orthogonally confirmable due to their very small magnitude of change. For statistical analysis of differential expression between given two groups, a moderated T-test with Benjamini-Hochberg Multiple Testing Correction was used. For statistical multiple comparison analysis of differential expression, 2-way ANOVA with age and sex main effects was performed, followed by Student-Newman-Keuls (SNK) pairwise post-hoc test. Visualizations of hierarchical clustering and principal component analysis (PCA) were performed in Strand NGS (Version 3.1, Bangalore, India).
Assembly: mm10
Supplementary files format and content: Tab-delimited text file contains raw FPKM values for each sample.

source_name: RPE-choroid eyecups

extract_protocol:
Eyecup harvesting was performed within the 9:00-11:00 am time window for all experimental groups. The purification of cell-specific, ribosomally loaded RNA from eyecup tissue from Tam-induced Aldh1l1-cre/ERT2+; NuTRAP+ mice was achieved following an established protocol, with slight modifications40,42,50. Individual eyecups were placed in 1.5 ml tubes containing 150 µl ice-cold homogenization buffer (50 mM Tris, pH 7.4; 12 mM MgCl2; 100 mM KCl; 1% NP-40; 1 mg/ml sodium heparin; 1 mM DTT) supplemented with 100 µg/ml cycloheximide (#C4859-1ML, Millipore Sigma), 0.5 mM spermidine (#05292-1ML-F, Millipore Sigma), 200 units/ml RNaseOUT Recombinant Ribonuclease Inhibitor (#10777019; Thermo Fisher), and 1X cOmplete, EDTA-free Protease Inhibitor Cocktail (#11836170001; Millipore Sigma). Each eyecup was homogenized using plastic pestle and cordless motor (two pulses of 5 sec duration/each). After pooling homogenates from 4 eyecups (4 eyecups/2 mice = biological replicate), each sample was taken up to 1ml final volume with homogenization buffer and gently resuspended. After centrifugation (10,000 x g, 10 min, at 4° C), 100 µl of the supernatant of each sample was saved as input. The remaining supernatant was transferred to a 2 ml round-bottom tube and incubated with 5 µg/µl of anti-GFP antibody (ab290; Abcam) at 4°C with end-over-end rotation overnight. Dynabeads Protein G for Immunoprecipitation (#10003D; Thermo Fisher) were washed three times in 1 ml ice-cold low-salt wash buffer (50 mM Tris, pH 7.5; 12 mM MgCl2; 100 mM KCl; 1% NP-40; 100 μg/ml cycloheximide; 1mM DTT). After removal of the last wash, the homogenate/antibody mixture was transferred to the 2 ml round-bottom tube containing the washed Protein-G Dynabeads and incubated at 4°C overnight with end-over-end rotation. Magnetic beads were collected using a DynaMag-2 magnet. Beads were then washed three times with 1 ml of high-salt wash buffer (50mM Tris, pH 7.5; 12mM MgCl2; 300mM KCl; 1% NP-40; 100μg/ml cycloheximide; 1mM DTT). Following the last wash, 350 µl of Buffer RLT (Qiagen) supplemented with 3.5 µl 2-β mercaptoethanol was added directly to the beads and incubated with mixing on a ThermoMixer (Eppendorf) for 10 minutes at room temperature. The beads were magnetically separated and the supernatant containing the target bead-bound ribosomes and associated RNA was transferred to a new tube. 350 µl of 100% ethanol was added to the tube (“positive” fraction: enriched in translatome associated to EGFP-tagged ribosomes) and then loaded onto a RNeasy MinElute column. RNA was isolated using RNeasy Mini Kit (#74104, Qiagen), according to manufacturer’s instructions. RNA was quantified with a Nanodrop One spectrophotometer (Thermo Fisher Scientific) and its quality assessed by HSRNA ScreenTape with a 4150 TapeStation analyzer (Agilent Technologies, Santa Clara, CA).
The NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L; New England Biolabs Inc., Ipswich, MA) was used with 25 ng of total RNA for the preparation of strand-specific sequencing libraries from TRAP-isolated RNA samples (input and positive fraction) according to manufacturer's instructions40. Briefly, polyA containing mRNA was purified using oligo-dT attached magnetic beads. mRNA was chemically fragmented and cDNA synthesized. For strand-specificity, the incorporation of dUTP instead of dTTP in the second strand cDNA synthesis does not allow amplification past this dUTP with the polymerase. Following cDNA synthesis, each product underwent end repair process, A tailing and adapter ligation. The cDNA products were further purified and enriched using PCR to make the final library for sequencing. Library sizing was performed with HS RNA ScreenTape (#5067-5579; Agilent Technologies). The libraries for each sample were pooled at 4 nM concentration and sequenced using an Illumina NovaSeq S4 system (PE150) at the OMRF Clinical Genomics Facility.

value: RPE-choroid eyecups

value: RPE Cells

value: Aldh1l1-cre/ERT2+; NuTRAP+

tag: fraction

value: Positive

value: NEBNext Ultra II Directional RNA Library Prep Kit for Illumina


title: THP1, UL135stopvirus, undifferentiated, day5, rep2


title: THP1, WTvirus, undifferentiated, day6, rep2


title: I_3T_13

value: Embryonic Kindey Cells

value: transfected


title: MCF7 E2 10 nM 30 min Rep2

description:
LGA_3_2
There are 4 raw data files (L001-L004) per each read end (R1 and R2)

value: E2 10 nM 30 min


title: 2168_2169_Pos


title: I_3T_1


title: THP1, WTvirus, differentiated, day5.5, rep2

value: peripheral blood

value: monocyte

value: differentiated


title: MCF7 E2 10 nM 30 min Rep1

description:
LGA_3_1
There are 4 raw data files (L001-L004) per each read end (R1 and R2)

value: Mammary gland, epithelial

value: WT


title: 2168_2169_Input

value: Input


title: THP1, WTvirus, undifferentiated, day5.5, rep2

value: undifferentiated


title: I_2T_9


title: THP1, WTvirus, undifferentiated, day5, rep2


title: I_2T_5


title: 2138_2151_Input


title: THP1, WTvirus, undifferentiated, day3, rep2


title: 2170_2171_Input


title: MCF7 Vehicle Rep2

description:
LGA_1_2
There are 4 raw data files (L001-L004) per each read end (R1 and R2)

value: Vehicle


title: 2126_2137_Pos


title: MCF7 E2 10 nM 5 min Rep1

description:
LGA_2_1
There are 4 raw data files (L001-L004) per each read end (R1 and R2)

value: E2 10 nM 5 min


title: I_2T_17


title: THP1, uninfected, differentiated, day8, rep2

value: mock


title: I_2T_1


title: I_2T_13


title: 2126_2137_Input

value: RPE-choroid eyecups

value: RPE Cells


title: GILZ TG LPS_3

city: Saarbruecken

first: Thierry

middle: M.

last: Legroux

state: Saarland

address: Campus Saarbrücken

department: Pharmaceutical biology

institute: Saarland University

description:
DESeq2_GILZ TG vs. WT LPS.txt
DESeq2_GILZ KO vs. GILZ TG LPS.txt
DESeq2_GILZ TG_CO vs. LPS.txt

data_processing:
Raw reads were demultiplexed and subjected to quality control through FastQC v0.11.2. Read processing was performed with grape-nf pipeline (v1.1.3) using Nextflow (v20.10.0) and mapped to GRCm38/mm10 assembly. The counts obtained after alignment were used to analyze differential expression using DESeq2 v1.40.2.
All analyses were conducted in the R programming language. DESeq2 analysis revealed differentially expressed genes (DEGs, p < 0.05) in the comparisons of GILZ KO vs. WT, GILZ TG vs. WT, and GILZ KO vs. GILZ TG under both untreated and LPS-treated conditions.
Assembly: GRCm38/mm10
Supplementary files format and content: tab-delimited text-files include TPM values for each sample.
Supplementary files format and content: tab-delimited text-files include DESeq2 data for all comparisons.

source_name: bone-marrow derived macrophages

growth_protocol: The bone marrow from femur and tibia was flushed out with media and passed through a 100 µm cell strainer before erythrocyte lysis in hypotonic buffer (155 mM NH4Cl, 10 mM KHCO3, 1 mM Na2EDTA) at 37°C for 3 min. Cells were cultured for differentiation (6 days) to BMMs in a humidified incubator at 37°C and 5% CO2 in RPMI-1640 cell culture media supplemented with 10% FBS, 1% 200mM L-glutamine, 100 U/ml penicillin/streptomycin, and 50ng/ml of murine M-CSF (BMM media). Experiments were performed in BMM media.

extract_protocol:
RNA was isolated using the High Pure RNA Isolation Kit (Roche, #11828665001) according to the manufacturer’s protocol and stored at ‑80°C. All RNA samples used for further preparation had an RNA integrity number > 9 according to the analysis in a 2100 Bioanalyzer (Agilent) using the RNA 6000 Nano Kit (Agilent, #5067-1513)
Libraries were prepared from 500 ng RNA. Poly(A) enrichment was performed on the input total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, #E7490) according to the manufacturer's instructions. The cDNA library preparation was conducted with the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, #E7420) as recommended by the supplier. In brief, first- and second-strand cDNA synthesis was performed, followed by adapter ligation and PCR amplification of the final library (12 cycles). PCR cleanup was performed using Agencourt AM‑Pure XP beads (Beckmann Coulter, #A63881). Libraries were sequenced for 1x 75 nt on a NextSeq500 (Illumina) sequencer.

treatment_protocol: 500,000 BMMs (1ml/well) were seeded in a 12-well plate and treated for 4h with LPS (100ng/ml, Invivogen) on the next day.

value: female

value: bone-marrow derived macrophages

value: GILZ TG

value: 100 ng/ml LPS

tag: treatment time

value: 4 h


title: I_1T_9

value: Embryonic Kindey Cells

value: transfected


title: 2113_2101_Input

value: Aldh1l1-cre/ERT2+; NuTRAP+

value: NEBNext Ultra II Directional RNA Library Prep Kit for Illumina


title: 2113_2101_Pos

value: Positive


title: MCF7 Vehicle Rep1

description:
LGA_1_1
There are 4 raw data files (L001-L004) per each read end (R1 and R2)

value: Mammary gland, epithelial

value: WT


title: THP1, uninfected, undifferentiated, day8, rep2

value: peripheral blood

value: monocyte

value: undifferentiated


title: GILZ TG LPS_1


title: I_1T_5


title: THP1, uninfected, differentiated, day6, rep2

value: mock

value: differentiated


title: MCF7 E2 10 nM 5 min Rep2

description:
LGA_2_2
There are 4 raw data files (L001-L004) per each read end (R1 and R2)

value: E2 10 nM 5 min


title: GILZ TG LPS_2

value: female

value: bone-marrow derived macrophages


title: 2138_2151_Pos

value: RPE-choroid eyecups

value: RPE Cells


title: 2111_2112_Pos


title: I_1T_17

value: Embryonic Kindey Cells


title: THP1, uninfected, undifferentiated, day6, rep2


title: GILZ KO LPS_3

description:
DESeq2_GILZ KO vs. WT LPS.txt
DESeq2_GILZ KO vs. GILZ TG LPS.txt
DESeq2_GILZ KO_CO vs. LPS.txt

value: GILZ KO

value: 100 ng/ml LPS

value: 4 h


title: THP1, WTvirus, undifferentiated, day1, rep2


title: 2111_2112_Input

value: Aldh1l1-cre/ERT2+; NuTRAP+

value: Input

value: NEBNext Ultra II Directional RNA Library Prep Kit for Illumina


title: THP1, uninfected, differentiated, day5.5, rep2

value: peripheral blood


title: GILZ KO LPS_2


title: I_1T_13

value: transfected


title: 2107_2108_Pos

value: Positive


title: THP1, uninfected, undifferentiated, day5.5, rep2

value: monocyte

value: mock

value: undifferentiated


title: I_1T_1


title: THP1, uninfected, undifferentiated, day5, rep2


title: GILZ KO LPS_1

value: female


title: WT LPS_3

description:
DESeq2_GILZ KO vs. WT LPS.txt
DESeq2_GILZ TG vs. WT LPS.txt
DESeq2_WT_CO vs. LPS.txt

value: bone-marrow derived macrophages

value: WT


title: THP1, uninfected, undifferentiated, day3, rep2


title: C_8M_5


title: 2105_2106_Pos

value: RPE-choroid eyecups

value: RPE Cells


title: WT LPS_2


title: THP1, uninfected, undifferentiated, day1, rep2


title: C_8M_17

value: Embryonic Kindey Cells


title: WT LPS_1

value: 100 ng/ml LPS


title: C_8M_13


title: THP1, UL138stopvirus, differentiated, day8, rep1

value: differentiated


title: 782_760_Input

value: Aldh1l1-cre/ERT2+; NuTRAP+


title: THP1, UL138stopvirus, undifferentiated, day8, rep1


title: GILZ TG control_2

description:
DESeq2_GILZ TG vs. WT CO.txt
DESeq2_GILZ KO vs. GILZ TG CO.txt
DESeq2_GILZ TG_CO vs. LPS.txt

value: GILZ TG

value: untreated


title: 765_675_Pos

value: NEBNext Ultra II Directional RNA Library Prep Kit for Illumina


title: C_8M_1


title: THP1, UL138stopvirus, differentiated, day6, rep1

value: peripheral blood


title: C_7M_9


title: 765_675_Input

value: Input


title: THP1, UL138stopvirus, undifferentiated, day6, rep1

value: monocyte


title: C_8M_9

value: mock


title: C_7M_5


title: 2107_2108_Input


title: C_7M_17


title: GILZ KO control_3

description:
DESeq2_GILZ KO vs. WT CO.txt
DESeq2_GILZ KO vs. GILZ TG CO.txt
DESeq2_GILZ KO_CO vs. LPS.txt

value: GILZ KO


title: GILZ KO control_2

value: female


title: THP1, UL138stopvirus, differentiated, day5.5, rep1


title: 2105_2106_Input


title: 762_763_Pos

value: Positive


title: 762_763_Input


title: THP1, UL138stopvirus, undifferentiated, day5.5, rep1

value: undifferentiated


title: 782_760_Pos

value: RPE-choroid eyecups


title: 616_761_Pos

value: RPE Cells


title: C_7M_1

value: Embryonic Kindey Cells


title: GILZ KO control_1

value: bone-marrow derived macrophages


title: C_7M_13


title: THP1, UL138stopvirus, undifferentiated, day5, rep1


title: GILZ TG control_3


title: 616_761_Input


title: THP1, UL138stopvirus, undifferentiated, day3, rep1


title: WT control_3

description:
DESeq2_GILZ KO vs. WT CO.txt
DESeq2_GILZ TG vs. WT CO.txt
DESeq2_WT_CO vs. LPS.txt

value: WT


title: WT control_2


title: 592_595_Pos

value: Aldh1l1-cre/ERT2+; NuTRAP+


title: GILZ TG control_1

value: GILZ TG


title: C_6M_9


title: C_6M_5


title: 592_595_Input


title: WT control_1

value: untreated


title: THP1, UL138stopvirus, undifferentiated, day1, rep1


title: 532_533_Pos

value: NEBNext Ultra II Directional RNA Library Prep Kit for Illumina


title: C_6M_17


title: THP1, UL135stopvirus, differentiated, day8, rep1

value: peripheral blood

value: differentiated


title: control lymph node

data_processing:
Sequencing was demultiplexed, the barcodes were identified and the genes were counted using the CellRanger 3.1 pipeline (https://www.10xgenomics.com/support/software/cell-ranger/release-notes/cr-release-notes)
Assembly: mm10
Supplementary files format and content: tab seperated values and matrix files

source_name: inguinal lymph node

extract_protocol:
Lymph nodes were digested in collagenase and Dnase and DCs were collected by FACS sorting for Lin- Cd11c+ MHCIIhi cells
Libraries were constructed using 10x Genomics default protocols following the Chromium 3' next gem gene expression v3.1

value: inguinal lymph node

value: Dendritic Cells

value: Wildtype


title: THP1, UL135stopvirus, undifferentiated, day8, rep1

value: monocyte


title: C_6M_13

value: mock


title: 532_533_Input

value: Input


title: THP1, UL135stopvirus, differentiated, day6, rep1


title: C_6M_1


title: 365_364_Pos

value: RPE-choroid eyecups

value: Positive


title: THP1, UL135stopvirus, undifferentiated, day6, rep1

value: undifferentiated


title: Tumor draining lymph node 2

value: Tumor-draining lymph node  (inguinal)

value: 4 weeks following induction

value: 4-hydroxy-tamxifen tumor induction

value: BRafCA, PtenloxP, Tyr::CreERT2


title: C_5M_9

value: Embryonic Kindey Cells


title: C_5M_5


title: Non Draining lymph node 1

source_name: Non-draining lymph node (axillary)

value: Non-draining lymph node (axillary)


title: Tumor draining lymph node 1


title: THP1, UL135stopvirus, undifferentiated, day5.5, rep1

value: peripheral blood


title: C_5M_17


title: C_5M_13


title: VE_caMPRA_plasmid_DNA

city: Boston

first: Janet

last: Song

state: Massachusetts

address: 3 Blackfan St

institute: Boston Children's Hospital

data_processing:
Sequencing data from the plasmid DNA and cDNA libraries described above were processed with cutadapt to remove adapters (Martin, 2011).
Barcodes were extracted using UMI-tools (Smith et al., 2017)
Reads were mapped using bwa mem (Li and Durbin, 2009) and assigned to caMPRA probes using featureCounts (Liao et al., 2014).
The 10bp barcode were clustered to recover barcodes with small sequencing errors using the multiplexed version of the UMI-tools directional method.
Barcodes for plasmid and cDNA samples were normalized to a barcode per million format to remove bias due to sequencing coverage. Each cDNA barcode was normalized to the barcode count in the plasmid pool, and log2 transformed. Only barcodes that were found in the plasmid pool and all 5 cDNA replicate pools were used in the analysis. Enhancer activity was assessed with the XXXX test at 5% FDR.
Assembly: hg19
Supplementary files format and content: xlsx file of caMPRA results for HARs, VEs, and CNEs
Supplementary files format and content: xlsx file of random mutagenesis caMPRA results for HARs
Library strategy: MPRA

source_name: Neuro2A/N2A cells (ATCC, CCL-131)

growth_protocol: Neuro2A/N2A cells (ATCC, CCL-131) were grown in 10% fetal bovine serum, Dulbecco’s Modified Eagle Medium, and 1% Penicillin-Streptomycin in a 5% CO2 incubator at 37C.

extract_protocol:
Cell pellets were washed with 1x PBS, and mRNA was extracted using the Dynabead mRNA Direct kit (Thermo Fisher, cat #61012), according to the manufacturer’s instructions.
mRNA was reverse transcribed using Superscript VILO Master Mix with EZ DNase (Thermo Fisher, cat #11766050). caMPRA barcodes were extracted using PCR amplification with primers containing illumina adapters for both the cDNA and plasmid pools and sent out for 150bp sequencing using Hiseq instruments at Psomagen (Rockville, MD).

treatment_protocol: Molecular Inversion Probes (MIPs) targeting HARs, VEs, and CNEs were designed using the MIPgen program (Boyle et al., 2014) and manufactured as a pool by CustomArray (Bothell, WA). Target genomic sequences were captured from DNA from sample NA12878 (Coriell Institute), and the caMPRA assay then was conducted as previously described (Girskis et al., 2021). Briefly, the captures were cloned into modified pMPRA1 vector (Addgene, 49349). The only modification to the pMPRA1 vector was the addition of an AsiSI restriction site used for inserting our captured DNA fragments. The luciferase gene and minimal promoter (pMPRA_donor2, Addgene, 49353) was then cloned into the pMPRA1_Capture construct pool. For random mutagenesis, we performed error-prone PCR using the GeneMorph II Random Mutagenesis Kit (Agilent, cat #200550) on the amplified, captured sequences. N2A cells were grown as adherent cultures in 15 cm plates. Then, 15ug of MPRA constructs were transfected into cells with Lipofectamine LTX reagent (Thermo Fisher, A12621). The cells were harvested 24 or 72 hr post-transfection. Five replicates were performed per condition.

value: Neuro2A/N2A cells (ATCC, CCL-131)


title: VE_caMPRA_D3_rep5


title: Littermate Controls [LMF3]

city: Fort Collins

first: Devin

last: Wahl

state: Colorado

address: 1582 Campus Delivery, Moby Reception

institute: Colorado State University

data_processing:
Adapters trimmed & file QC by Fastp
Reads aligned with STAR and counting with LiBiNorm
Differential expression analyzed with DESeq2
Assembly: mm10

source_name: Hippocampus

extract_protocol:
RNA for polyA RNA-seq extracted using Trizol and Zymo spin columns.
PolyA libraries were prepared according to manufacturer's instructions using the Illumina TruSeq kit

value: Hippocampus

tag: condition

value: Littermate Controls

value: female


title: THP1, UL135stopvirus, undifferentiated, day3, rep1

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using trimmomatic (parameters - LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36)
Trimmed sequence reads were mapped to concatenate GRCh38/CMV using STAR
Read count extraction and normalization were performed using subread
Assembly: Concatenated Homo_sapiens.GRCh38/Human herpesvirus 5 strain TB40/E clone TB40-BAC4
Supplementary files format and content: Microsoft Excel worksheet contains raw counts for each sample
Supplementary files format and content: Microsoft Excel worksheet contains RPKM values for each sample

source_name: peripheral blood

extract_protocol:
Total RNA was extracted with the Zymo Duet Quick-DNA/RNA Miniprep kit. Manufacturer protocols were followed for column isolation of total RNA, DNAse treatment, and RNA cleaning and concentration.
cDNA libraries were prepared with an Illumina TruSeq Stranded mRNA kit and a KAPA Dual-Indexed Adaper kit. The NGS library was quantified with a qPCR-based KAPA Library Quantification kit. Manufacturer protocols were followed.

value: monocyte


title: C_5M_1

value: mock


title: THP1, UL135stopvirus, undifferentiated, day5, rep1


title: Non Draining lymph node 2

value: Dendritic Cells


title: THP1, UL135stopvirus, undifferentiated, day1, rep1


title: VE_caMPRA_D3_rep4


title: VE_caMPRA_D3_rep3


title: VE_caMPRA_D3_rep2


title: C_4T_9

value: transfected


title: Littermate Controls [LMF1]


title: rTg4510+3TC [DM3]

value: male


title: C_4T_5


title: Littermate Controls [LMM3]


title: THP1, UL135stopvirus, differentiated, day5.5, rep1

value: differentiated


title: THP1, WTvirus, differentiated, day8, rep1